Oligomeric compounds having nitrogen-containing linkages

ABSTRACT

Novel compounds and libraries of compounds based on nitrogen atoms that are joined together with spanner groups include &#34;letters&#34; i.e., functional groups, that are attached to the nitrogen atoms, to the spanner groups or to both the nitrogen atoms and the spanner groups to render the compounds and libraries of such compounds with diverse properties.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of the following U.S.applications Ser. No. 08/180,124, filed Jan. 11, 1994 now U.S. Pat. No.5,783,682; Ser. No. 08/039,979, filed Mar. 30, 1993 now abandoned; Ser.No. 08/039,846, filed Mar. 30, 1993 now abandoned; Ser. No. 08/040,933,filed Mar. 31, 1993 now abandoned; Ser. No. 08/040,903, filed Mar. 31,1993 now U.S. Pat. No. 5,386,023; and Ser. No. 08/040,526, filed Mar.31, 1993 now U.S. Pat. No. 5,489,677. Each of the foregoing arecontinuations-in-part of PCT/US92/04294, filed May 21, 1992, and of U.S.Ser. No. 07/903,160, filed Jun. 24, 1992 now abandoned, which arecontinuations-in-part of U.S. Ser. No. 07/703,619, filed May 21, 1991now U.S. Pat. No. 5,378,825, which is a continuation-in-part of U.S.Ser. No. 07/566,836, filed Aug. 13, 1990 now U.S. Pat. No. 5,223,618,and U.S Ser. No. 07/558,663, filed Jul. 27, 1990 now U.S. Pat. No.5,138,045. Each of these patent applications are assigned to theassignee of this application and are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the design, synthesis and application ofoligomeric compounds containing monomeric units that each include anitrogen atom plus a. "spanner," i.e. a group of atoms that spansbetween and connects adjacent nitrogen atoms. The monomeric units areconnected together in linear or cyclic arrays. The monomeric units aresubstituted, via substitution on the nitrogen atom and/or substitutionon the spanner with a tethered or untethered functional group. Theoligomers are synthesized having either a random or a predefinedsequences of units. Randomization can be effected independently at thefunctional functional group. The oligomers are synthesized having eithera random or a predefined sequences of units. Randomization can beeffected independently at the functional group or at the spanners. Thefunctional group on each of the monomeric units provides for binding ofthe oligomeric structures to proteins, nucleic acids, lipids and otherbiological targets. In preferred embodiments, the compounds of theinvention act as inhibitors of enzymes such as phospholipase A₂ : asinhibitors of pathogens such as virus, mycobacterium, bacteria (gramnegative and gram positive), protozoa and parasites; as inhibitors ofligand-receptor interactions such as PDGF (platelet derived growthfactor), LTB4 (leukotriene B4), IL-6 and complement C5_(A) ; asinhibitors of protein/protein interactions including transcriptionfactors such as p50 (NF_(kappa) B protein) and fos/jun; and for theinhibition of cell-based interactions including ICAM induction (usinginducers such as IL1-β, TNF and LPS). In other preferred embodiments,the compounds of the invention are used as diagnostic reagents,including diagnostic reagents in the tests for each of the above notedsystems, and as reagents in assays and as probes.

BACKGROUND OF THE INVENTION

Traditional processes of drug discovery involve the screening of complexfermentation broths and plant extracts for a desired biological activityor the chemical synthesis of many new compounds for evaluation aspotential drugs. The advantage of screening mixtures from biologicalsources is that a large number of compounds are screened simultaneously,in some cases leading to the discovery of novel and complex naturalproducts with activity that could not have been predicted otherwise. Thedisadvantages are that many different samples must be screened andnumerous purifications must be carried out to identify the activecomponent, often present only in trace amounts. On the other hand,laboratory syntheses give unambiguous products, but the preparation ofeach new structure requires significant amounts of resources. Generally,the de novo design of active compounds based on the high resolutionstructures of enzymes has not been successful.

In order to maximize the advantages of each classical approach, newstrategies for combinatorial unrandomization have been developedindependently by several groups. Selection techniques have been usedwith libraries of peptides (see Geysen, H. M., Rodda, S. J., Mason, T.J., Tribbick, G. & Schoofs, P. G., J. Immun. Meth. 1987, 102, 259-274;Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C.T. & Cuervo, J. H., Nature, 1991, 354, 84-86; Owens, R. A., Gesellchen,P. D., Houchins, B. J. & DiMarchi, R. D., Biochem. Biophys. Res.Commun., 1991, 181, 402-408), nucleic acids (see Wyatt, J. R., et al.,Proc. Natl. Acad. Sci. USA, 1994, 91, 1356-1360; Ecker, D. J., Vickers,T. A., Hanecak, R., Driver, V. & Anderson, K., Nucleic Acids Res., 1993,21, 1853-1856) and nonpeptides (see Simon, R. J., et al., Proc. Natl.Acad. Sci. USA, 1992, 89, 9367-9371; Zuckermann, R. N., et al., J. Amer.Chem. Soc., 1992, 114, 10646-10647; Bartlett, Santi, Simon, PCTWO91/19735; and Ohlmeyer, M. H., et al., Proc. Natl. Acad. Sci. USA,1993, 90, 10922-10926). The techniques involve iterative synthesis andscreening of increasingly simplified subsets of oligomers. Monomers orsub-monomers that have been utilized include amino acids and nucleotidesboth of which are bi-functional. Utilizing these techniques, librarieshave been assayed for activity in either cell-based assays, or forbinding or inhibition of purified protein targets.

A technique, called SURF (Synthetic Unrandomization of RandomizedFragments), involves the synthesis of subsets of oligomers containing aknown residue at one fixed position and equimolar mixtures of residuesat all other positions. For a library of oligomers four residues longcontaining three monomers (A, B, C), three subsets would be synthesized(NNAN, NNBN, NNCN, where N represents equal incorporation of each of thethree monomers). Each subset is then screened in a functional assay andthe best subset is identified (e.g. NNAN). A second set of libraries issynthesized and screened, each containing the fixed residue from theprevious round, and a second fixed residue (e.g. ANAN, BNAN, CNAN).Through successive rounds of screening and synthesis, a unique sequencewith activity in the assay can be identified. The SURF technique isdescribed in Ecker, D. J., Vickers, T. A., Hanecak, R., Driver, V. &Anderson, K., Nucleic Acids Res., 1993, 21, 1853-1856. The SURF methodis further described in PCT patent application WO 93/04204, the entiredisclosure of which is herein incorporated by reference.

The combinatorial chemical approach that has been most utilized to date,utilizes an oligomerization from a solid support using monomeric unitsand a defined connecting chemistry, i.e. a solid support monomerapproach. This approach has been utilized in the synthesis of librariesof peptides, peptoids, carbamates and vinylogous peptides connected byamide or carbamate linkages or nucleic acids connected by phosphatelinkages as exemplified by the citations in previous paragraphs above.The mixture of oligomers (pool or library) is obtained from the additionof a mixture of activated monomers during the coupling step or from thecoupling of individual monomers with a portion of the support (beadsplitting) followed by remixing of the support and subsequent splittingfor the next coupling. In this monomeric approach, each monomeric unitwould carry a tethered letter, i.e., a functional group for interactionwith the target. A further coupling chemistry that allows for theinsertion of a tethered letter, at a chemically activated intermediatestage is referred to as the sub-monomer approach.

The diversity of the oligomeric pool is represented by the inherentphysical properties of each monomer, the number of different monomersmixed at each coupling, the physical properties of the chemical bondsarising from the connecting chemistry (the backbone), the number ofcouplings (length of oligomer), and the interactions of the backbone andmonomer chemistries. Taken together these interactions provide a globalshape for each individual molecule.

There remains a need in the art for molecules which have fixedpreorganized geometry that matches that of a target such as proteins andenzymes, nucleic acids and lipids. The backbone of such molecules shouldbe rigid with some flexibility and easy to construct in solution or viaautomated synthesis on solid support. We have developed certain nitrogencoupled chemistries that we utilized to prepare a class of compounds werefer to as "oligonucleosides." We have described these compounds inprevious patent applications including published PCT applications WO92/20822 (PCT US92/04294) and WO 94/22454 (PCT US94/03313). Thesechemistries included amine linkages, hydroxylamine linkages, hydrazinolinkages and other nitrogen based linkages. We have now found that thesesame linkages can be utilized to prepare linear and cyclized oligomericcompounds that carry functional groups thereon that are capable ofinteracting with a variety of target structures including proteins andenzymes, nucleic acids, lipids and other target molecules.

OBJECTS OF THE INVENTION

It is an object of the invention to provide oligomeric compounds fordiagnostic, research, and therapeutic use.

It is a further object of the invention to provide oligomeric compoundswherein functional groups are coupled to at least some of the monomericunits of the oligomeric compounds via nitrogen atoms in the monomericunit.

It is yet another object of the invention to provide methods forcombinatorial synthesis of libraries of oligomeric compounds.

It is yet another object of the invention to provide libraries ofcombinatorized compounds.

These and other objects will become apparent to persons of ordinaryskill in the art from a review of the present specification and theappended claims.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that mimic, modulate orotherwise interact with various target molecules including proteins andenzymes, nucleic acids and lipids. In certain embodiments, the compoundscontain one or more selected functional groups for interactions with thetarget molecule. At least a portion of the compounds of the inventionhas structure I: ##STR1## wherein: each R_(N) is, independently, H,--T--L, C₂ -C₁₀ alkyl or substituted alkyl, C₂ -C₁₀ alkenyl orsubstituted alkenyl, C₂ -C₁₀ alkynyl or substituted alkynyl, C₄ -C₇carbocylo alkyl or alkenyl, an ether having 2 to 10 carbon atoms and 1to 4 oxygen or sulfur atoms, a polyalkyl glycol, or C₇ -C₁₄ aralkyl orsubstituted aralkyl; a nitrogen, sulfur or oxygen containingheterocycle; and where the substitutents groups are selected fromhydroxyl, alkoxy, alcohol, benzyl, phenyl, nitro, thiol,n thioalkoxy,halogen, or alkyl, aryl, alkenyl, or alkynyl groups;

each Q is, independently, N--R_(N), O, S, SO, SO₂ or (CH₂)_(m) where mis 1-5;

k is zero or 1;

each A is, independently, R_(S) --X(T--L)--R_(S) ; N--R_(N) ; C(O); asingle bond; (CH₂)_(m) where m is 1-5; or CR¹ R_(N) ;

each R_(S) is, independently, a single bond or alkyl having 1 to about12 carbon atoms;

each T is, independently, a single bond, a methylene group or a grouphaving structure II:

    --[CR.sup.1 R.sup.2 ].sub.n --B--[CR.sup.1 R.sup.2 ].sub.o --[D].sub.p --[N(R.sub.N)].sub.q --

where:

D is C(O), C(S), C(Se), C(R¹)(NR³ R⁴), CH₂ R¹, CHR¹ R², or NR³ R⁴ ;

B is a single bond, CH═CH, C.tbd.C, O, S or NR⁴ ;

each R¹ and R² is independently selected from the group consisting ofhydrogen, alkyl or alkenyl having 1 to about 12 carbon atoms, hydroxy-or alkoxy- or alkylthio-substituted alkyl or alkenyl having 1 to about12 carbon atoms, hydroxy, alkoxy, alkylthio, amino and halogen;

R³ and R⁴, independently, are H, --T--L, alkyl having 1 to about 10carbon atoms; alkenyl having 2 to about 10 carbon atoms; alkynyl having2 to about 10 carbon atoms; aryl having 7 to about 14 carbon atoms;heterocyclic; a conjugate molecule; or

R³ and R⁴, together, are cycloalkyl having 3 to about 10 carbon atoms orcycloalkenyl having 4 to about 10 carbon atoms;

n and o, independently, are zero to 5;

q is zero or 1;

p is zero to about 10;

each L is, independently, C₂ -C₁₀ alkyl or substituted alkyl, C₂ -C₁₀alkenyl or substituted alkenyl, C₂ -C₁₀ alkynyl or substituted alkynyl,C₄ -C₇ carbocylo alkyl or alkenyl or C₇ -C₁₄ aralkyl or sustututedaralkyl, and where the substitutents groups are selected from hydroxyl,alkoxy, alcohol, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, oralkyl, aryl, alkenyl, or alkynyl groups; an ether having 2 to 10 carbonatoms and 1 to 4 oxygen or sulfur atoms; a polyalkyl glycol; a nitrogen,sulfur or oxygen containing heterocycle; a metal coordination group; aconjugate group; halogen; hydroxyl (OH); thiol (SH); keto (C═O);carboxyl (COOH); amide (CONR); ethers; thioethers; amidine (C(═NH)NRR);guanidine (NHC(═NH)NRR); glutamyl CH(NRR)(C(═O)OR); nitrate (ONO₂);nitro (NO₂); nitrile (CN); trifluoromethyl (CF₃); trifluoromethoxy(OCF₃); O-alkyl; S-alkyl; NH-alkyl; N-dialkyl; O-aralkyl; S-aralkyl;NH-aralkyl; amino (NH₂); azido (N₃); hydrazino (NHNH₂); hydroxylamino(ONH₂); sulfoxide (SO); sulfone (SO₂); sulfide (S--); disulfide (S--S);silyl; a nucleosidic base; an amino acid side chain; a carbohydrate; adrug; or group capable of hydrogen bonding;

each X is, independently, N or CH, or

X and T, together, form an aromatic moiety.

Preferred nitrogen blocking groups of the invention includetert-butoxycarbonyl, sulfenyltriphenyl and phthaloyl nitrogen protectinggroup.

The compounds of the invention generally are prepared by couplingpreselected bifunctional synthons under conditions effective to form theabove-noted structures. In certain embodiments, the compounds of theinvention are prepared by intermolecular reductive coupling of, forexample, a hydrazine moiety on a first synthon with an aldehyde moietyon a second synthon. In other embodiments, the compounds of theinvention are prepared by coupling a carbocentric radical on a firstsynthon with, for example, a radical acceptor moiety on a secondsynthon. In further embodiments, the compounds are prepared through anucleophilic alkylation wherein a nucleophilic moiety on a first synthondisplaces a leaving group on a second synthon.

The present invention is further directed to libraries of compounds.Preferable libraries include combinatorialized of compounds wherein theindividual members of libraries comprise compounds of the structure:

    N(R.sub.N)--[(CH.sub.2).sub.m --Q--N(R.sub.N)].sub.z --N(R.sub.N)

wherein

Q is N, O or (CH₂)_(m) ;

each R_(N) is a member of a group of letters;

m is 1 to 5; and

z is 2 to 100.

In a more preferred size range of the members of the libraries, z aboveis 2 to 25. In still other embodiments of the invention z is 2 to 10. Aneven more preferred range is wherein z is 2 to 5.

A first preferred group of letters, i.e. the variable R_(N) above,include aryl or substituted aryl letters. A further preferred group ofletters include amino acid side chain letters. A further preferred groupof letters include aliphatic, substituted aliphatic, aromatic orsubstituted aromatic letters.

In certain preferred libraries of compounds the invention, theindividual members of the libraries are formed from linked units derivedfrom intermediates of the structure:

    R.sub.X --(CHφ).sub.0-1 --(CH.sub.2).sub.0-5 --(CHφ).sub.0-1 --R.sub.Y

wherein:

φ is a letter, a tethered letter or H, and provided that in thosecompounds having a single φ, then φ is a letter or a tethered letter;and in those compounds having multiple φs, then at least one φ is aletter or a tethered letter and the remaining are either H, a letter ortethered letter;

R_(X) is aldehyde, ketone, halide, acid or acid halide;

R_(Y) is N₃, NO₂, N-bg, ON-bg, NφN-bg or SO₂ N-bg; and

bg is a nitrogen blocking group or a solid phase support.

In further preferred libraries of compounds the invention, theindividual members of the libraries are formed from linked units derivedfrom intermediates of the structure:

    R--(CR.sub.2 R.sub.3).sub.1-10 --R.sub.4 --NR.sub.6

wherein:

R is aldehyde, ketone, halide, acid or acid halide;

R₂ and R₃ are H, alkyl, substituted alkyl, aryl, substituted aryl,aralkyl, substituted aralkyl, heterocycle, moiety as found in α-positionof amino acids, halogen, amine, substituted amine, hydroxy, alkoxyl,substituted alkoxyl, SH, or substituted thioalkoxyl;

R₄ is O, CH₂, CR₂ R₃, NH, NR₅ or SO₂

R₅ is alkyl, substituted alkyl, aryl or substituted aryl; and

R6 is phthaloyl, H₂, N₂, O₂, H Acetyl, diAcetyl, methyleneamino, or anamino protecting group.

BRIEF DESCRIPTION OF THE DRAWINGS

The numerous objects and advantages of the present invention may bebetter understood by those skilled in the art by reference to theaccompanying figures, in which:

FIG. 1 shows solid phase and solution phase processes for synthesis ofhydrazino-linked compounds according to the invention;

FIG. 2 shows solid phase and solution phase processes for synthesis ofamino-linked compounds according to the invention;

FIG. 3 shows further solid phase and solution phase processes forsynthesis of amino-linked compounds according to the invention;

FIG. 4 shows solid phase and solution processes for synthesis ofhydroxylamino-linked compounds according to the invention;

FIG. 5 shows a synthetic scheme for synthesis of hydroxylamino-linkedcompounds according to the invention by radical coupling methodology;

FIG. 6 shows solid phase processes for synthesis of duplex, hairpin,stem-loop, and cyclic hydroxylamino-linked compounds according to theinvention;

FIG. 7 shows solid phase processes for synthesis of duplex, hairpin,stem-loop, and cyclic amino-linked compounds according to the invention;

FIG. 8 shows solid phase and solution processes for synthesis of certainintermediate compounds for the preparation of libraries of compoundsaccording to the invention;

FIGS. 9a and 9b shows solid phase and solution processes for a firstround of synthesis for preparing libraries of compounds according to theinvention;

FIG. 10 shows solid phase and solution processes for a second round ofsynthesis for preparing libraries of compounds according to theinvention;

FIG. 11 shows solid phase and solution processes for a third round ofsynthesis for preparing libraries of compounds according to theinvention;

FIG. 12 shows solid phase and solution processes for a fourth round ofsynthesis for preparing libraries of compounds according to theinvention; and

FIG. 13 shows solid phase and solution processes for a fifth round ofsynthesis for preparing libraries of compounds according to theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the invention are shown by Structure I above. In StructureI, two repeating unit or monomeric unit are illustrated. Higherpolymeric compounds of the invention would, of course, includeadditional monomeric unit of the same structure. Each of the monomericunits includes at least one nitrogen atom therein. This nitrogen atomcan be one of various moieties that include a nitrogen atom as anintegral part of the moiety. Preferred as such nitrogen based moiety areamine, hydroxylamine, hydrazine and sulfonamide moieties. A functionalgroup, i.e. a "letter," can be covalent bonded to the nitrogen atom ofthe nitrogen moiety to introduce a point of functionality at theparticular monomeric unit. When so functionalized, the nitrogen atom ofthe monomeric unit would be a tertiary nitrogen. Alternately aparticular monomeric unit might include a "null" in place of thefunctional group. In one instance, this is accomplished by having thenitrogen atom as a secondary nitrogen, i.e. R_(N) is H in the aboveStructure I.

Each monomeric unit can also be viewed as including a "spanner" moietythat connects between adjacent nitrogen atoms. Together, the spannerportions and the nitrogen atoms are covalently bonded into an oligomericbackbone. Thus, the spanner groups are, in essences, bi-functional innature and alternate with the nitrogen atoms to form a backbone thatincludes one or more functional groups projecting there from. Thebackbone can be linear or it can cyclized back on itself to form acyclic polymeric compound. It is understood that in speaking of a"nitrogen atom" and a "spanner group" in one context and of nitrogencontaining moieties in a further context, certain atoms, e.g. the oxygenatom of a hydroxylamino group or the second nitrogen of a hydrazinogroup, of the nitrogen based moieties may for the sake of description bein a first instance part of the nitrogen based moieties and in a secondinstance be part of the spanner group.

The nitrogen atoms of the backbone, besides being linked together by thespanner groups, serve also as the primary site for connecting thefunctional groups that impart "functional" properties to the oligomericcompounds of the invention. By varying these functionalgroups--diversity is incorporated into the compounds of the invention.Except when they are located on the ends of the oligomeric compounds ofthe invention or they carry a "null" group thereon, the nitrogen atomsare trivalent in nature--that is they are connected to at least twospanner groups (one on either side) and to one functional group. In somepreferred embodiments of compounds of the invention there will be from 2to about 100 such spanner groups. In still other preferred compounds ofthe invention, there will be from 2 to 25 such spanner groups. A morepreferred range is from 2 to 10 such groups. An even more preferredrange is from 2 to 5 such groups.

In addition to linking the nitrogen atoms of the backbone together in anoligomeric structure, a particular spanner group can also carry afunctional group thereon. Thus the functional groups can be locatedeither on the nitrogen atoms of the backbone, on the spanner groups oron both the nitrogen atoms and the spanner groups. The functional groupsare attached to the nitrogen atoms of the backbone or to the spannergroups with or without intervening tethers.

The functional groups appended to these oligomeric compounds of theinvention can be of various structures that impart particularinteractive properties to the oligomeric compounds. These functionalgroups can effect interactions of at least the following types:hydrogen-bond donors and acceptors, ionic, polar, hydrophobic, aromatic,electron donors and acceptors, pi bond stacking or metal binding.

The functional groups are also referenced as "letters." The use of suchterminology reflects the fact that the different functional groups onthe monomeric units of the compounds of the invention are positioned insequences (either predetermined or by random selection) much likeletters of the alphabet--thus the term "letter." These letters can be"reactive" or "non-reactive." By reactive, it is meant that they willinteract with a target molecule in some manner (that need not but can bepredefined). By non-reactive, it is meant that they are not designed toprimarily interact with a target molecule, and in fact while they mayinteract with the target molecule, the primary purpose of thenon-reactive moieties are to impart other properties to the moleculesuch as, but not necessary limited to, effecting up-take, distribution,metabolism or identification.

A first preferred group of functional groups according to the inventioninclude but are not limited to aromatic moieties and substitutedaromatic moieties, halogen (Cl, Br, F, I), hydroxyl (OH), thiol (SH),keto (C═O), carboxyl (COOH), amide (CONR), ethers, thioethers, amidine(C(═NH)NRR), guanidine (NHC(═NH)NRR), glutamyl CH(NRR)(C(═O)OR), nitrate(ONO₂), nitro (NO₂), nitrile (CN), trifluoromethyl (CF₃),trifluoromethoxy (OCF₃), O-alkyl, S-alkyl, NH-alkyl, N-dialkyl,O-aralkyl, S-aralkyl, NH-aralkyl, amino (NH₂), azido (N₃), hydrazino(NHNH₂), hydroxylamino (ONH₂), sulfoxide (SO), sulfone (SO₂), sulfide(S--), disulfide (S--S), silyl, heterocyclic, alicyclic, carbocyclic,conjugate groups and metal coordination groups. Preferred substituentsinclude substituted and unsubstituted aryl and aralkyl having from 6 to20 carbons atoms, halogens, alcohols and ethers (OR), thiols andthioethers (SR), amines (NRR), amidines [C(═NH)NRR], guanidines[NHC(═NH)NRR], aldehydes (CH═O), acids [C(═O)OH], esters [C(═O)OR],amides [C(═O)NRR], glycine [CH(NH₂)(C(═O) OH)], purine and pyrimidineheterocycles.

In further preferred embodiments of the invention, the reactivefunctionalities used as letters, suitable for use in the practice ofthis invention include, but are not limited to, substituted orunsubstituted heterocyclic compounds, such as substituted orunsubstituted heterocycloalkyls; amino containing groups, such asheterocycloalkylamines, polyalkylamines, imidazoles, imidazole amides,alkylimidazoles; substituted or unsubstituted aldehydes; substituted orunsubstituted acids; substituted or unsubstituted amides; substituted orunsubstituted ketones; substituted or unsubstituted ethers; substitutedor unsubstituted esters; substituted or unsubstituted aralkylaminohaving from about 6 to about 20 carbon atoms, aminoaralkylamino havingfrom about 6 to about 20 carbon atoms, alkyloxyaryl compounds, orallyloxyaryl compounds.

The functional groups or letters are attached to the nitrogen atoms ofthe backbone or to the spanner groups with or without interveningtethers. Tethers as used in the context of this invention are bivalentgroups that have a first end for covalently bonding to the nitrogenatoms of the backbone or to the spanner group and a second end capableof binding a letter. Such tethers can be used to position "letters" inspace with respect to the backbone or to link letters to the nitrogenatoms of the backbone wherein the letter itself does not include anactive moiety capable of covalently bonding to a nitrogen of thebackbone. A particularly preferred group of compound useful as tethersinclude, but are not limited to, C₂ -C₁₀ alkyl, C₂ -C₁₀ alkenyl, C₂ -C₁₀alkynyl, C₄ -C₇ carbocylo alkyl or alkenyl, heterocycles, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, polyalkylglycols and C₇ -C₄ aralkyl groups.

Amine functional groups of the invention can include amines of all ofthe above alkyl, alkenyl and aryl groups including primary and secondaryamines and "masked amines" such as phthalimide. Amines of this inventionare also meant to include polyalkylamino compounds and aminoalkylaminessuch as aminopropylamines and further heterocycloalkylamines, such asimidazol-1, 2, or 4-yl-propylamine.

Other reactive functionalities suitable for practicing the inventioninclude, without limitation, compounds having thiol (SH), aldehyde(C═O), or alcohol (OH) functionalities.

Heterocycles, including nitrogen heterocycles, suitable for use asfunctional groups include, but are not limited to, imidazole, pyrrole,pyrazole, indole, 1H-indazole, α-carboline, carbazole, phenothiazine,phenoxazine, tetrazole, triazole, pyrrolidine, piperidine, piperazineand morpholine groups. A more preferred group of nitrogen heterocyclesincludes imidazole, pyrrole, and carbazole groups. Imidazole groups areespecially preferred.

Further preferred heterocycles include the purines and pyrimidines.Purines and pyrimidines suitable for use as functional groups includeadenine, guanine, cytosine, uridine, and thymine, as well as othersynthetic and natural nucleobases such as xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudo uracil), 4-thiouracil, 8-halo, amino, thiol, thioalkyl, hydroxyland other 8-substituted adenines and guanines, 5-trifluoromethyl andother 5-substituted uracils and cytosines, 7-methylguanine. Furtherpurines and pyrimidines include those disclosed in U.S. Pat. No.3,687,808, those disclosed in the Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, and those disclosed by Englisch, et al., AngewandteChemie, International Edition 1991, 30, 613.

For the purposes of this specification, in the context of the inventionand in reference to the above Structure I, alkyl, alkenyl, and alkynylgroups include but are not limited to substituted and unsubstitutedstraight chain, branch chain, and alicyclic hydrocarbons, includingmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, nonadecyl, eicosyl and other higher carbon alkylgroups. Further examples include 2-methylpropyl, 2-methyl-4-ethylbutyl,2,4-diethylpropyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl,6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,2-ethylhexyl and other branched chain groups, allyl, crotyl, propargyl,2-pentenyl and other unsaturated groups containing a pi bond,cyclohexane, cyclopentane, adamantane as well as other alicyclic groups,3-penten-2-one, 3-methyl-2-butanol, 2-cyanooctyl, 3-methoxy-4-heptanal,3-nitrobutyl, 4-isopropoxydodecyl, 4-azido-2-nitrodecyl,5-mercaptononyl, 4-amino-1-pentenyl as well as other substituted groups.

Further, in the context of this invention, a straight chain compoundmeans an open chain compound, such as an aliphatic compound, includingalkyl, alkenyl, or alkynyl compounds; lower alkyl, alkenyl, or alkynylas used herein include but are not limited to hydrocarbyl compounds fromabout 1 to about 6 carbon atoms. A branched compound, as used herein,comprises a straight chain compound, such as an alkyl, alkenyl, alkynyl,which has further straight or branched chains attached to the carbonatoms of the straight chain. A cyclic compound, as used herein, refersto closed chain compounds--that is, a ring of carbon atoms, such as acyclic aliphatic or aromatic compound. The straight, branched, or cycliccompounds may be internally interrupted (i.e., alkylalkoxy orheterocyclic compounds). In the context of this invention, internallyinterrupted means that the carbon chains may be interrupted withheteroatoms such as O, N, or S; however, if desired, the carbon chainmay have no heteroatoms.

For the purposes of this specification, in the context of the inventionand in reference to the above Structure I, aryl groups include but arenot limited to substituted and unsubstituted aromatic hydrocarbylgroups. Aralkyl groups include but are not limited to groups having botharyl and alkyl functionality, such as benzyl and xylyl groups. Preferredaryl and aralkyl groups include, but are not, limited to, phenyl,benzyl, xylyl, naphthyl, tolyl, pyrenyl, anthracyl, azulyl, phenethyl,cinnamyl, benzhydryl, and mesityl. These can be substituted orunsubstituted. It is particularly preferred that if substituted, thesubstitution be meta to the point of attachment of the substitution arylor aralkyl compound to the backbone or tether connecting to the backbonesince such meta substitution isolates electronic effects of thesubstituent from the reactive functionality used to attached thearomatic moiety to the backbone or tether.

Such compounds as noted above may be substituted or unsubstituted. Inthe context of this invention, substituted or unsubstituted, means thatthe compounds may have any one of a variety of substituents, inreplacement, for example, of one or more hydrogen atoms in the compound,or may have no substituents. Typical substituents for substitutioninclude for example, but are not limited to, substituted with hydroxyl,alkoxy, alcohol, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, oralkyl, aryl, alkenyl, or alkynyl groups.

Metal coordination groups according to the invention include but are notlimited to hydroxamic acids, catecholamide, acetylacetone,2,2'-bipyridine, 1,10-phenanthroline, diacetic acid,pyridine-2-carboxamide, isoalkyldiamine, thiocarbamate, oxalate, glycyl,histidyl and terpyridyl. Other metal coordination groups are known, asfor example see Mellor, D. P., Chemistry of Chelation and ChelatingAgents in International Encyclopedia of Pharmacology and Therapeutics,Section 70, The Chelation of Heavy Metals, Levine, W. G. Ed., PergamonPress, Elmford, N.Y., 1979.

Non-reactive functionalities used as letters, such as groups thatenhance pharmacodynamic properties, include groups that improve uptake,enhance resistance to enzymatic or chemical degradation, and/orstrengthen sequence-specific interaction with a target molecule.Non-reactive functionalities may also enhance pharmacokineticproperties, in the context of this invention, such groups improveuptake, distribution, metabolism or excretion. Non-reactivefunctionalities include, but are not limited to, alkyl chains,polyamines, ethylene glycols, steroids, polyamides, aminoalkyl chains,amphipathic moieties, points for reporter group attachment, andintercalators attached to any of the preferred sites for attachment, asdescribed above.

Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include PEG groups, cholesterols, phospholipids,biotin, phenanthroline, phenazine, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes.

A number of functional groups can be introduced into compounds of theinvention in a blocked form and subsequently de-blocked to form a final,desired compound. In general, blocking groups render chemicalfunctionality inert to specific reaction conditions and can be appendedto and removed from such functionality in a molecule withoutsubstantially damaging the remainder of the molecule. See, e.g., Greenand Wuts, Protective Groups in Organic Synthesis, 2d edition, John Wiley& Sons, New York, 1991. For example, amino groups can be blocked asphthalimido groups or as 9-fluorenylmethoxycarbonyl (FMOC) groups andcarboxyl groups can be protected as acetyl groups. Representativehydroxyl protecting groups are described by Beaucage, et al.,Tetrahedron 1992, 48, 2223. Preferred hydroxyl protecting groups areacid-labile, such as the trityl, monomethoxytrityl, dimethoxytrityl,trimethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthine-9-yl (MOX).

Solid supports according to the invention include controlled pore glass(CPG), oxalyl-controlled pore glass (see, e.g., Alul, et al., NucleicAcids Research 1991, 19, 1527), TentaGel Support--anaminopolyethyleneglycol derivatized support (see, e.g., Wright, et al.,Tetrahedron Letters 1993, 34, 3373) or Poros--a copolymer ofpolystyrene/divinylbenzene.

Oligomeric compounds of the invention can be synthesized with thesequence of letters predetermined or random. Thus in certain preferredembodiments, the sequence of letters is a predetermined sequence. Infurther preferred embodiments, the sequence of letters is random. Ineven further preferred embodiments, the sequence is modulated betweenfixed and random. This is especially useful, as for example, in certaincombinatorial'strategies such as the above referenced SURF strategy.

A further advantage of this invention is the ability to synthesizeoligomeric compounds that, in addition to or in place of variability inthe sequences of the letters, have an asymmetric sequence of spannerunits. Stated otherwise, the spanner units can also vary within anoligomeric structure. This is easily accomplished by using differentcompounds that eventually become incorporated as spanner units.

One preferred method of synthesizing the compounds of the invention isvia a solution phase synthesis. A further preferred method ofsynthesizing the compounds of the invention is a solid phase synthesis.

The letters are attached to their respective monomeric units, either tothe nitrogen atom of the backbone of the monomeric unit or to thespanner group of the monomeric unit. These functional groups thusprovide diverse properties ("diversity") to the resulting oligomericcompounds. Such diversity properties include hydrogen-bond donors andacceptors, ionic moieties, polar moieties, hydrophobic moieties,aromatic centers, electron-donors and acceptors, pi bond stacking andmetal binding. Together, the properties of the individual repeatingunits contribute to the uniqueness of the oligomer in which they arefound. Thus, a library of such oligomers would have a myriad ofproperties, i.e., "diversity." Collectively, the properties of therepeating units that form an oligomer contribute to the uniqueness ofsuch an oligomer and impart certain characteristics thereto forinteraction with protein, lipid, cellular, enzymatic or nucleic acidtarget sites.

The oligomeric compounds of the invention can be prepared having eitherpreselected sequences or sequences determined via combinatorialstrategies. One useful combinatorial strategy is the above-noted SURFstrategy, which is disclosed and claimed in U.S. patent application Ser.No. 749,000, filed Aug. 23, 1991, and PCT Application US92/07121, filedAug. 21, 1992, both of which are commonly assigned with thisapplication. The entire disclosure of these applications are hereinincorporated by reference.

Illustrative of the SURF strategy is a 2'-O-methyl oligonucleotidelibrary (see, Ecker et. al., ibid.) shown in Table I, below. Table Idescribes the selection of a 2'-O-methyl oligonucleotide for binding toan RNA hairpin. The K_(D) 's, i.e., the binding constants, weredetermined by gel shift. "X" is used to indicate the position beingvaried and underlining is used to indicate positions that become fixedduring successive iterations of the SURF strategy.

                  TABLE I                                                         ______________________________________                                                   K.sub.D (mM)                                                       Subsets      X = A   X = C     X = G X = T                                    ______________________________________                                        Round 1                                                                         NNNNXNNNN 22 10  >100 >100                                                    Round 2                                                                       NNNNCNXNN >10 4  >10 >10                                                      Round 3                                                                       NNXNCNCNN >10 0.5  >10 >10                                                    Round 4                                                                       NNCXCNCNN >10 0.15  >10 >10                                                   Round 5                                                                       NNCCCXCNN 0.08  >1 0.4 >1                                                     Round 6                                                                       NNCCCACXN 0.05  >0.5 0.08 >0.5                                                Round 7                                                                       NXCCCACAN >0.1 >0.1 0.03  >0.1                                                Round 8                                                                       NGCCCACAX 0.05 0.02  0.05 0.04                                                Round 9                                                                       XGCCCACAC  0.03 0.05 0.02 0.01                                              ______________________________________                                    

This SURF strategy has not been previously used for libraries exceptthose that employ naturally-occurring nucleotides as phosphodiesters orphosphorothioates as monomeric units. Other combinatorial strategieshave only been previously used for libraries that employ amino acids asmonomeric units.

One advantage of the present invention is that the simple design ofrepeating units enables combining rational drug design with screeningmechanisms for thousands of compounds. This is achieved by using thecompounds of the invention in a combinatorial techniques such as theSURF strategies.

The oligomeric compounds of the invention can be used in diagnostics,therapeutics and as research reagents and kits. They can be used inpharmaceutical compositions by including a suitable pharmaceuticallyacceptable diluent or carrier. In preferred embodiments, the compoundsof the invention act as inhibitors of enzymes such as phospholipase A₂ :as inhibitors of pathogens such as virus, mycobacterium, bacteria (gramnegative and gram positive), protozoa and parasites; as inhibitors ofligand-receptor interactions such as PDGF (platelet derived growthfactor), LTB4 (leukotriene B4), IL-6 and complement C5_(A) ; asinhibitors of protein/protein interactions including transcriptionfactors such as p50 (NF_(kappa) B protein) and fos/jun; and for theinhibition of cell-based interactions including ICAM induction (usinginducers such as IL1-β, TNF and LPS). In other preferred embodiments,the compounds of the invention are used as diagnostic reagents for eachof the above noted biological entities, and as reagents in assays and asprobes.

The compounds of the invention generally are prepared by couplingpreselected bifunctional synthons under conditions effective to formcompounds having structure I. In certain embodiments, compounds of theinvention are prepared by intermolecular reductive coupling. In otherembodiments, compounds of the invention are prepared by intermolecularradical addition reactions. In further embodiments, compounds areprepared by nucleophilic displacement. In each of these embodiments,free amino groups in the resulting linkage can be furtherfunctionalized. For example, the nucleophilic amino group can be reactedwith a group having structure R_(L) --T--L, thereby displacing the R_(L)leaving group and forming a covalent --N--T--L linkage.

In the reductive coupling methods, compounds having structure I areformed by coupling synthons having structures III and IV: ##STR2##wherein: R_(N1) and R_(N2) are, independently, amine protecting groups,or a group comprising: [N(R_(N))--Q--A--CH₂ --]_(r) where r is 1-100, orR_(N1) and R_(N2) together, form an amine protecting group; and

R_(A1) and R_(A2) are, independently, carbonyl protecting groups, or agroup comprising: [N(R_(N))--Q--A--CH₂ --]_(r) where r is 1-100, orR_(A1) and R_(A2), together, form a carbonyl protecting group.

Each R_(N) is independently, H, --T--L, C₂ -C₁₀ alkyl or substitutedalkyl, C₂ -C₁₀ alkenyl or substituted alkenyl, C₂ -C₁₀ alkynyl orsubstituted alkynyl, C₄ -C₇ carbocylo alkyl or alkenyl, an ether having2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, a polyalkylglycol, or C₇ -C₁₄ aralkyl or substituted aralkyl; a nitrogen, sulfur oroxygen containing heterocycle; and where the substitutents groups areselected from hydroxyl, alkoxy, alcohol, benzyl, phenyl, nitro, thiol,nthioalkoxy, halogen, or alkyl, aryl, alkenyl, or alkynyl groups.

The radical addition reactions can be divided into two steps. The firststep involves generation of an initial radical, which undergoes thedesired reaction. The second step involves removal of the radical fromthe reaction before the occurrence of an intervening, undesired reactionsuch as cross coupling.

In certain embodiments, the compounds of the invention are prepared byproviding a donor synthon having structure V and an acceptor synthonhaving structure VI where R_(B) is a radical generating group,generating a carbocentric radical at the --CH₂ --R_(B) position, andthen forming an intermolecular linkage by reacting radical-bearing donorsynthon V with acceptor synthon VI. Radical generating groups accordingto the invention include I, OC(S)O--C₆ H₅, Se--C₆ H₅, OC(S)O--C₆ F₅,OC(S)O--C₆ Cl₅, OC(S)O--(2,4,6--C₆ Cl₃), Br, NO₂, Cl, OC(S) S--Me,OC(S)O--(p-CH₄ F), bis-dimethylglyoximatopyridine cobalt, OC(S)C₆ H₅,OC(S)SCH₃, OC(S)-imidazole, and OC(O)O-pyridin-2-thione. ##STR3##

The nucleophilic displacement (alkylation) reactions involve reacting afirst synthon VII bearing a leaving group, R_(L), with a second synthonVIII bearing a nucleophilic nitrogen moiety under conditions effectiveto displace the leaving group and form the above-identified linkages.##STR4## Leaving groups according to the invention include chloro,fluoro, bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl,benzenesulfonyl, methylsulfonyl (mesylate), p-methylbenzenesulfonyl(tosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate),trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl,imidazolesulfonyl, and 2,4,6-trichlorophenyl groups.

The linkages of the invention can be formed by selecting aformyl-derivatized compound (e.g., structure III) as an upstream synthonand an amino-derivatized compound (e.g., structure IV) as a downstreamsynthon.

Formyl-terminated compounds such as structure III can be formed viaseveral synthetic pathways. One preferred method utilizes a radicalreaction of the corresponding xanthate-terminated compound. The xanthatecompound is treated with 2,2'-azobisisobutrylonitrile (AIBN), andtributyltin styrene in toluene. Subsequently, the styrene derivative ishydroxylated and cleaved to furnish a formyl group. Alternately,formyl-terminated compounds can be synthesized from a cyano-terminatedcompound by techniques well known in the art. Terminal formyl groups canbe blocked in a facile manner, for example, utilizingO-methylaminobenzenthiol as a blocking group. The formyl blocking groupcan be deblocked with silver nitrate oxidation.

An alternate method of preparing formyl-terminated compounds employstosylation of a terminal hydroxyl group, which on iodination followed bycyanation with KCN in DMSO will furnish a nitrile. Reduction withDIBAL-H gives the desired formyl-terminated compound. In yet anothermethod, a terminal C═C bond is oxidized with OsO₄ and cleavage of theresulting diol with NaIO₄ gives the desired formyl functionality.

Hydroxylamino terminated compounds such as those having structure IV(Q═O) can be prepared by treating the corresponding hydroxyl compoundwith N-hydroxyphthalimide, triphenylphosphine anddiethylazodicarboxylate under Mitsunobu conditions to provide anO-phthalimido derivative. The free hydroxylamino compound can begenerated in quantitative yield by hydrazinolysis of the O-phthalimidoderivative.

Hydrazino-terminated compounds such as those having structure IV (Q═NH)can be prepared by treating hydroxyl-terminated compounds with tosylchloride in pyridine to give an O-tosylate derivative. Treatment ofbenzylcabazide with O-tosylate will furnish a benzylcarbazidederivative, which on hydrogenation provides the free hydrazino moietyfor reductive coupling.

Amino-terminated compounds such as those having structure IV (Q═CH₂) canbe synthesized by treating the corresponding hydroxyl-terminatedcompound with Ph₃ P, CBr₄ and LiN₃ according to the procedure of Hata,et al., J. Chem. Soc. Perkin 1 1980, 306, to furnish a terminal azide.Reduction of the azido group with tributyltin hydride provides thedesired amino functionality.

Coupling of structures III and IV then is effected to furnish a dimericunit having an imine or oxime linkage. This linkage then is reduced insitu with NaCNBH₃ to furnish a --C--N-- linked unit.

Oligomers containing a uniform backbone linkage can be synthesized usingCPG-solid support and standard synthesizing machines such as PerkinElmer Applied Biosystems Inc. 380B and 394 and Milligen/Biosearch 7500and 8800s. The initial monomer is attached, via an appropriate linker,to a solid support such as controlled pore glass or polystyrene beads.In sequence specific order, each new monomer (e.g., structure III or IV)is attached either by manual manipulation or by the automatedsynthesizer system. In the case of a methylenehydrazine linkage (Q═N),the repeating nucleoside unit can be of two general types: a linearstructure with a protected aldehydic function at one end and aC-hydrazinomethyl group at the opposite end, or a structure bearing aterminal hydrazino group and a protected C-formyl group. In each case,the conditions that are repeated for each cycle to add the subsequentbase include: acid washing to remove the terminal aldehydo protectinggroup; addition of the next molecule with a methylenehydrazino group toform the respective hydrazone connection; and reduction with any of avariety of agents to afford the desired methylene-hydrazine linked CPG-or polystyrene-bound structure. One such useful reducing agent is sodiumcyanoborohydride.

A preferred method is shown in FIG. 1. This method utilizes a solidsupport to which a linear molecule having a protected aldehyde or analdehyde precursor at its terminal end is attached. The terminalaldehyde can be suitably protected with various groups, such asdescribed by Greene and Wuts in Protective Groups in Organic Synthesis,John Wiley & Sons, Inc., 1991, pp 175-223. In one preferred method, thealdehyde group is protected with N,N'-diphenyl imidazolidine, which canbe cleaved with aqueous HCl as described by Giannis, et al. Tetrahedron1988, 44, 7177. 2,3-Dihydro-1,3-benzo-thiazole is yet another preferredprotecting group for aldehyde functionality and is cleaved by AgNO₃ atneutral pH (see, e.g., Trapani, et. al., Synthesis 1988, 84). Morepreferably, a terminal vinyl group is oxidized with OsO₄ and cleavedwith NaIO₄ to yield a free aldehydo group.

A bifunctional synthon having a protected aldehydo group at one end (themasked coupling end) and a hydrazino group at the opposite end (thereactive coupling end) can be coupled under acidic conditions with alinear aldehyde attached to the solid support. The intermediatehydrazone then is reduced with NaBH₃ CN to furnish a hydrazino linkageattached to the solid support.

Subsequently, bisalkylation of the hydrazino moiety via an appropriatehalide or aldehyde provides a N,N-substituted hydrazine linked to thesolid support. Thereafter, the cycle can be repeated by the addition ofbifunctional synthon under acidic conditions, reduction, and alkylationof hydrazine moiety to create a polymeric molecule of a desired sequenceconnected by one or more substituted hydrazino linkages. In somepreferred embodiments of this invention, the final unit utilized forcoupling can bear an ionic linkage to provide water solubility for suchmolecules.

One preferred process employs an aldehyde-protected synthon attached tothe solid support. Attachments can be effected via standard proceduresas described by R. T. Pon in Protocols For Oligonucleotides And Analogs,Chapter 24, Agrawal, S., ed., Humana Press, Totowa, N.J., 1993.

As an alternative, a solution phase synthesis of substituted hydrazinolinked linear molecules can be accomplished via hydroxyl protectedsynthons, such as shown in FIG. 1 (R_(Z) =hydroxyl protecting group orsolid support) utilizing a t-butyl diphenylsilyl group.

A further method of synthesizing N-substituted hydroxylamine linkedlinear molecules is depicted in FIG. 4 (L_(S) =a linker attached tosolid support, or a protecting group, such as t-butyldiphenylsilyl).This method also employs a solid support to which a linear moleculehaving an O-phthalimido group at its terminal end is attached. A furtherbifunctional unit that has an aldehyde functionality at the coupling endand an O-phthalimido group at the growing end is utilized as the middleblock via repeating cycles. The synthesis of polymeric structures can bestopped by use of a terminating unit that bears a hydroxyl protectinggroup rather than a phthalimido group. A wide variety of hydroxylprotecting groups can be employed in the methods of the invention. Ingeneral, protecting groups render chemical functionality inert tospecific reaction conditions, and can be appended to and removed fromsuch functionality in a molecule without substantially damaging theremainder of the molecule. Representative protecting groups aredescribed by Beaucage, et al., Tetrahedron 1992, 48, 2223.

The O-phthalimido group attached to the support is hydrazinolyzed withmethylhydrazine to generate a reactive O-amino group. Acid catalyzedcoupling of the resulting bifunctional unit provides an oxime linkedsupport. The oxime linkage can be reduced with NaBH₃ CN/acetic acid toyield a hydroxyl amino linkage, which is then alkylated with appropriatefunctionality. Alternately, the coupled unit can be treated with methylhydrazine and the coupling with bifunctional unit repeated until anoligomer of desired length is obtained. The multiple oxime linkages thuscreated can be reduced in one step utilizing NaBH₃ CN/AcOH to createfree O-amino groups, which can be further substituted uniformly withappropriate functionality.

In a similar manner, a solution phase synthesis of such polymericmolecules connected via substituted hydroxylamino linkages utilizes thecoupling/reduction/alkylation hydrazinolysis steps in a sequentialorder, starting with a hydroxyl protected molecule.

The radical-based methods of the invention generally involve "nonchain"processes. In nonchain processes, radicals are generated bystoichiometric bond homolysis and quenched by selective radical-radicalcoupling. It has been found that bis(trimethylstannyl)benzopinacolateand bis(tributylstannyl)benzopinacolate (see, e.g., ComprehensiveOrganic Synthesis: Ed. by B. M. Trost & J. Fleming, Vol. 4, pp760)--persistent radicals--can be used to enhance the radical-radicalcoupling and reduce cross-coupling. It will be recognized that apersistent radical is one that does not react with itself at adiffusion-controlled rate. Hillgartner, et al., Liebigs. Ann. Chem.1975, 586, disclosed that on thermolysis (about 80° C.) pinacolateundergoes homolytic cleavage to give the suspected persistent radical(Ph₂ C.sup.• OSnMe₃), which stays in equilibrium with benzophenone andthe trimethylstannyl radical (Me₃ Sn.sup.•) It is believed that the Me₃Sn.sup.• radical abstracts iodine from radical precursors such asiodo-terminated compounds having structure V to give radical-terminatedintermediates. The radicals then add to immino acceptors such asstructure VI to yield a --C--C--N-- linkage.

At high concentrations the initial radical can be trapped by couplingprior to addition, and at low concentrations the adduct radical canbegin to telomerize. It is believed that a three molar equivalent excessof pinacolate provides satisfactory results for such couplings. Theefficiency of radical reactions is highly dependent on the concentrationof the reagents in an appropriate solvent. Preferably, the reactionmixture contains benzene, dichlorobenzene, t-butylbenzene, t-butylalcohol, water, acetic acid, chloroform, dichloromethane, carbontetrachloride, or mixtures thereof. The solvent should contain acombined concentration of about 0.1 to about 0.4 moles/liter of radicalprecursor and acceptor, preferably about 0.1 to about 0.2 moles/liter.It has been found that best results are obtained using benzene solutionscontaining about 0.2 moles/liter of radical precursor and acceptor.

As exemplified in FIG. 5, the radical coupling of an oxime ether 31 asan acceptor with radical precursor 33 occurs in the presence ofbis(trimethylstannyl)benzopinacolate in refluxing benzene. The reactionis carried out under argon and a 35-50% isolated yield of the product isobtained after purification. The hydroxylamino linkages thus obtainedcan be alkylated with an appropriate functionality. Subsequently, thehydroxyl group is deblocked and treated with N-hydroxyphthalimide underMitsunobu conditions to yield an O-phthalimido derivative.Hydrazinolysis and formylation of the latter compound gives an oximeether functionality at the reactive end of the molecule. Therefore, aradical coupling cycle can be repeated with high chemoselectivity toyield an oligomer or polymeric unit linked via one or more substitutedhydroxylamino linkages. The chain elongation can be terminated at anypoint during the described method by avoiding the Mitsunobu reaction atthe hydroxyl function.

The desired method essentially can be transferred from solution to solidphase systems by utilizing an oxime unit linked to a support via alinker.

The radical coupling methodology also can employ a bifunctional unit, asdepicted in FIG. 5. Thus, coupling between an oxime linked to a supportand the bifunctional unit under the described conditions will provide ahydroxylamino linked molecule. This compound can be alkylated in astandard manner to yield a N-substituted molecule. Subsequently,deblocking of the phthalimido group with methyl hydrazine liberates afree O-amino group, which on treatment with formaldehyde gives aterminal oxime. The oxime can be used in another round of coupling withan iodo derivative. In this manner the synthesis is more convenient, dueto the Mitsunobu reaction prior to coupling. Radical coupling cycles canbe repeated as often as needed until a polymer of desired length isobtained. The elongation usually is terminated by using a last unit, asshown in FIG. 5, that bears a protected hydroxyl group. The foregoingprocedure is highly adaptable to solution phase chemistry in a similarmanner.

The synthesis of libraries of oligomeric compounds of the invention isillustrated in FIGS. 8-13. The individual compound species in theselibraries are generated via combinatorial methodologies. Illustrated inthese figures is the preparation of intermediates used for the synthesisof libraries of compounds of the invention and combinatorialmethodologies for synthesizing such libraries utilizing theseintermediates.

The libraries are prepared by general procedures that results innitrogen based combinatorial libraries. The active species of thelibraries are determined using a SURF deconvolution procedure. Bothsolution phase and solid phase synthesis are used to create thelibraries. Example 8 of this specification illustrates the generalcombinatorial synthesis and deconvolution procedures used to create thelibraries. Example 9 illustrates the synthesis of intermediates used inboth solution phase and solid phase synthesis of libraries. Creation ofa full library of compounds and determination of such active species viaa SURF deconvolution is illustrated in Example 10. Example 11illustrates the activation of solid phase support intermediates forattachment to controlled pore glass supports, i.e. CPG. Example 12illustrates the attachment of the activated intermediates on to CPGsolid support for use in solid phase possesses that parallel thesolution phase processes illustrated in Example 10. The solid phasesynthesis is effected in the same manner as the solution phase synthesiswith the exception that bead splitting is substituted for the solutionsplitting of the solution phase synthesis. Example 13 illustratesloading of solid phase intermediates onto organic resins.

Example 10 illustrates the preparation of libraries via Schiff's basealkylation whereas Example 14 illustrates the preparation of librariesvia alkylation reaction using halide intermediates. Example 15illustrates a further method for preparing the libraries via acylationusing acid halides intermediates. Example 16 illustrates alternatemethods for the preparation of "extenders," (that form the spanners orportions thereof). Examples 17 and 18 illustrate further "extenders."These are amino acid types and oxyamine acid types, respectively.

In FIGS. 8-13, the synthesis and combinatorialization of1,4,9,14,19-pentaaza-8,12,18-trioxanonadecane with nitrogens 1,4,9,14,19combinatorialized with four letters is illustrated. A total of 1024compounds are prepared in four sets of 256 compounds each. Four sets oflinear polyamine/oxyamines, compounds 122a-d, are formed in round 1.Each set has positions 1 through 5 (positions 1 and 5 are primarynitrogens where as positions 2, 3 and 4 are secondary oxyaminenitrogens) substituted in a combinatorial manner, i.e.combinatorialized, with equal amounts of the letters. The followingstructures identify the position numbers and the nomenclature numberingused in the FIGS. 8-13 and their accompanying examples.

    H--N.sup.1 --(CH.sub.2).sub.2 --N.sup.2 --(CH.sub.2).sub.3 O--N.sup.3 --(CH.sub.2).sub.3 O--N.sup.4 --(CH.sub.2).sub.3 O--N.sup.5 --H(position numbers)

    H--N.sup.1 --(CH.sub.2).sub.2 --N.sup.4 --(CH.sub.2).sub.3 O.sup.8 --N.sup.9 --(CH.sub.2).sub.3 O.sup.13 --N.sup.14 --(CH.sub.2).sub.3 O.sup.18 N.sup.19 --H                                     (nomenclature numbering)

For illustrative purposes aromatic letters, specifically benzyl,m-methylbenzyl, m-nitrobenzyl, and m-methoxybenzyl moieties, are used asletters. The precursors compounds for these letters as well as formultitudes of other such letters are commercially available from variouscommercial sources. Other letters, for example alkyl, alkenyl, alkynyl,amino acid side chains, nucleobases and the like, are utilized in thesame manner. As illustrated in the Figures, at the completion ofsynthesis of the library, each set has position 5 (a primary oxyaminenitrogen) substituted exclusively with a known one of the four letters.For illustrative purposes, as shown in the Figures, the letter selectedto be fixed is placed in the molecule last. This was selected asposition 5 for fixing in the first round synthesis of the libraries. Forthe illustrative compounds that have five combinatorial sites with onesite fixed, the iterative deconvolution process (SURF) requires foursubsequent rounds of synthesis to be performed to identify the mostactive molecules. Each round of synthesis is performed to allow theposition selected to be fixed as the last fixed position. Other positionselection approaches can be taken, e.g. the first selected position canbe fixed. For illustrative purposes, to fix a position last in rounds3-6, the acid labile sulfenyl triphenyl methyl moiety is utilized toprotect the designated nitrogen atom until combinatorilization of otherpositions and fixing of known positions is completed. Other protectivegroups can also be utilized.

The linear polyamine/oxyamines are prepared by two sets of sequentialreactions: submonomer addition of a letter to a secondary nitrogen andextension (elongation) of the chain via an extender to provide anothersecondary nitrogen for combinatorialization via submonomer chemistry(first step). The purpose of repeating these sequential sets ofreactions is to liberate/provide a reactive secondary amine (the nextposition to be combinatorialized) in the growing chain in the absence ofother reactive centers and to extend the molecular length (and thus thenumber of combinatorial positions) in the polyamine/oxyamine chain.

The key starting material for this particular combinatorial chemistrylibrary is1-(tert-butoxycarbonyl)-9-phthaloyl-1,4,9-triaza-8-oxa-decane, i.e.t-Boc--NH--(CH₂)₂ --NH--(CH₂)₃ --O--N-phthaloyl, 106. This same compoundcan also be used as a key starting material for preforming thecombinatorial synthesis on a solid support. This is illustrate whereBG=solid support, e.g. 1-(BG)-9-phthaloyl-1,4,9-triaza-8-oxa-decane,i.e. BG--NH--(CH₂)₂ --NH--(CH₂)₃ --O--N-phthaloyl, 109.

For the solution phase synthesis, the material 106 is protected at oneend (position 1) with the acid labile tert-butoxycarbonyl group (t-Boc)and the terminal oxyamine (position 3) is protected by a base labilephthaloyl group (acid dephthalyation can also be used if desired). Theinternal secondary oxyamine (position 2) is unprotected and availablefor submonomer chemistry as shown in compound 106. In addition, 106 withits internal secondary amine protected with a sulfenyltriphenylprotecting group is employed in preparations of latter rounds ofdeconvolution.

The basic chemistry employs three types of tertiary nitrogens, a primaryamine, a primary oxyamine and a secondary oxyamine, each of which isprotected with a suitable protecting group. For illustrative purposes,the protective groups selected for protecting these nitrogens aretert-butoxycarbonyl, a sufenyltriphenyl and a phthaloyl. Thetert-butoxycarbonyl (t-Boc) and the sulfenyltriphenyl [S(Ph)₃ ] moietiesare remove by various differential acid conditions and the phthaloylmoiety is typically removed with hydrazines (basic conditions) and withcertain acid conditions. For addition of letters, the monomer and thesubmonomer approaches are utilized. The submonomer approach requires theaddition of a letter intermediate to one of the tertiary nitrogens. Thiscan be accomplished by several chemistries including, but not limitedto, "Schiff's base reductive alkylation," alkylation, e.g., withalkyl-halides, and amide bond formation with acid, acid halides, esters,etc.

The Schiff's base reductive alkylation is described in Examples 9 and10. Aralkyl halide chemistry is described in Example 14 and acylationchemistry is described in Example 15. Moieties required for submonomerletter addition are aldehydes, ketones, primary and secondary alkylhalides, sulfonates, trifilates, diazonium salts, acids, acid halides,esters, etc. These same reactive moieties are employed to extend thechain when attached to an alkyloxyphalimide or a substitutedalkyloxyphalimide (both in monomer and submonomer approaches). Startingmaterials for these reaction are commercial chemical reagents availablefrom various commercial chemical supply houses. They can be used aspurchased without further modification.

The illustrative oligomeric compound having five site forcombinatorialization and four letters, is treated via submonomerchemistry to combinatorializes position 2 with four letters by a splitsolution procedure. Each letter is reacted separately with 106. Afterpurification, if needed, an equal molar amount of each pure compoundbearing a different letter is mixed together to provide a mixture offour compounds with position 2 combinatorialized with four letters.

Position 3 is deprotected (dephthaloylation with methylhydrazine),followed by reductive alkylation (Schiff's base formation and reductionof imino intermediate with NaCNBH₃) with a selected extender. Asillustrated in the figures, the selected extender isN-(3-hydroxypropionaldehyde)phthalimide, 105, i.e. OHC(CH₂)₂ O-NPhth. Avariety of other extenders can be employed including aldehydes, ketones,halides, acid halides and the like. Preferred representative extendersinclude:

    ______________________________________                                        OHCCHφ-N.sub.3                                                                        BrCHφCHφ-N.sub.3                                                                     ClCOCHφ-N.sub.3                                  OHCCHφ-NO.sub.2 BrCHφCHφ-NO.sub.2 ClCOCHφ-NO.sub.2                                      OHCCHφ-NPhth BrCHφCHφ-NPhth                                      ClCOCHφ-NPhth                                    OHCCHφO--NPhth BrCHφCHφO--NPhth ClCOCHφO--NPhth                                         OHCCHφNφ-NPhth BrCHφCHφNφ-                               NPhth ClCOCHφNφ-NPhth                        OHCCHφSO.sub.2 --NPhth BrCHφCHφSO.sub.2 --NPhth ClCOCHφS                               O.sub.2 --                                             NPhth                                                                     ______________________________________                                    

where Phth is phthalimide and φ is a letter (functional group), atethered letter or H; and provide that in those compounds having asingle φ, then φ is a letter or a tethered letter; and in thosecompounds having multiple φs, then at least one φ is a letter or atethered letter and the remaining are either H, a further letter ortethered letter. Other preferred representative extenders are of theformula:

    R.sub.X --(CHφ).sub.0-1 --(CH.sub.2).sub.0.5 --(CHφ).sub.0-1 --R.sub.Y

where:

Phth and φ are as defined above;

R_(X) is HCO, ketone, halide, CO-halide; and

R_(Y) is N₃, NO₂, NPphth, ONPhth, NφNPhth and SO₂ NPhth.

Halides in this instances are Br, Cl and I.

Modified extenders allows the addition of letters through the monomerapproach. A further group of preferred extenders, some of which includemodification such as side chains and the like, include compounds of thestructure:

    R--(CR.sub.2 R.sub.3).sub.1-10 --R.sub.4 --NR.sub.6

where:

R is OHC-- (aldehydes), OR₁ C-- (ketones) halogen, HO₂ C--, andhalogen-CO-- (acid halide);

R₂ and R₃ are H, alkyl, substituted alkyl, aryl, substituted aryl,aralkyl, substituted aralkyl, heterocycle, moiety as found in α-positionof amino acids, halogen, amine, substituted amines, hydroxy, alkoxyls,substituted alkoxyls, SH, and substituted thioalkoxyls;

R₄ is O, CH₂, CR₂ R₃, NH, NR₅ and SO₂,

R₅ is alkyl, substituted alkyl, aryl and substituted aryl;

R6 is phthaloyl, H₂, N₂, O₂, H Acetyl, diAcetyl, methyleneamino, or anamino protecting group; and

CR₂ R₃ are a chain of 3 atoms or more containing C, N, O, S and theirvarious oxidation states.

The reaction with the extender provides a reactive secondary nitrogenready for combinatorialization. The sequential set of reaction arerepeated again. This is continued until the desired length is obtained(for the number of combinatorial sites, for the length of molecule, forthe desired molecular weight, etc.). As illustrated in the examples,compound 117 (having four sites for combinatorialization) is extendedone additional time to provide five site for combinatorialization. Thenposition 1 is liberated with acid conditions. The resulting primaryamine is treated separately with each of the four letters. In this case,reduction of the intermediate imino (--CH═N--) moiety is not performeduntil the final step to allow a cleaner reactions when position 5 issubsequently fixed with each specific letter. The final set of reactionsfor the preparation of this particular library provides a fixed letterat position 5. In this case position 5 is not combinatorialized--it isnot a mixture of the four letters. Each set of the library has a knownletter at position 5. Thus four subsets of libraries, (mixtures ofcompounds) are obtained. This approach employed a split solution processutilizing SURF deconvolution (iterative screening).

Extension (elongation) of the chain provides another secondary nitrogenfor combinatorialization via submonomer chemistry. The purpose ofrepeating these sequential sets of reactions is to liberate/provide areactive secondary amine (the next position to be combinatorialized) inthe growing chain in the absence of other reactive centers and toextend, via an extender, the molecular length (and thus the number ofcombinatorial positions) in the polyamine/oxyamine chain. In addition toextending the length of the oligomeric compound, the extender could alsocarry a letter substituted in the molecule between the reactive group,e.g. aldehyde, and the protected amine, oxyamine or other nitrogenspecies. The extenders can be utilized with various chemistriesincluding the Schiff's base reductive alkylation (with aldehydes andketones) of Example 10, alkylation (with alkyl halides) of Example 14and with acylation (acid halides) following reduction of the amide bondof Example 15. Various types and mixtures of extenders can be used inthe elongation reaction.

EXAMPLE 1 Reductive Coupling

I. Solution phase Synthesis of an Oligomeric Molecule Linked ViaHydrazino Linkages (FIG. 1)

A. Synthesis of a `First Unit`,1-O-(t-butyldiphenylsilyl)-butyraldehyde-1-ol, 3 (R_(Z)=t-butyldiphenylsilyl (TBDPS), r=1)

A mixture of 4-penten-1-ol (10 mmol), t-butyldiphenylsilylchloride (12mmol), imidazole (25 mmol) and dry DMF (50 ml) is stirred at roomtemperature for 16 h under argon. The reaction mixture is poured intoice-water (200 ml) and the solution extracted with CH₂ Cl₂ (2×200 ml).The organic layer is washed with water (2×200 ml) and dried (MgSO₄). TheCH₂ Cl₂ layer is concentrated to furnish a gummy residue, which onpurification by silica gel chromatography gives silylated 4-penten-1-ol.The silylated compound is oxidized with OsO₄ (1 mmol) andN-methylmorpholine oxide (20 mmol) in diethyl ether (40 ml) and water(20 ml) at room temperature for 18 h. NaIO₄ (30 mmol) solution in water(2 ml) is added to the above solution and stirring is continued for 12h. The aqueous layer is extracted with diethyl ether (2×200 ml) andevaporation of combined organic layers gives crude aldehyde 3.

B. Synthesis of a `Bifunctional Units`, 4-Penten-1-hydrazinehydrochloride, 8, and Imidazolidine Derivative, 5

Treatment of 4-Penten-1-ol with tosylchloride in pyridine will furnishtosylated 6, which on treatment with benzylcarbazate indimethylacetamide as described in Example 1 of Ser. No. 08/039,979,filed Mar. 30, 1993, provides the carbazyl derivative 7. Hydrogenationwith Pd/C in MeOH/HCl provides the title compound 8 as hydrochloridesalt.

The aldehyde group of 3 is protected as N,N'-diphenylimidazolidinederivative utilizing the procedure of Giannis, et. al., Tetrahedron1988, 44, 7177, to furnish 4. Subsequently, 4 is treated with Bu₄ NF/THFto deblock the silyl protecting group. The hydroxyl group of the lattercompound is transformed into a hydrazino group via the two stepprocedure described above to yield title compound 5.

C. Synthesis of a `Terminal Unit`,3-O-(t-butyldiphenylsilyl)-1-(hydrazine)-propanol hydrochloride (11,R_(Z) =TBDPS, r=1).

The title compound is prepared from propane-1,3-diol, via selectivesilylation with t-butyl-diphenylsilylchloride, followed by treatmentwith benzylcarbazate and hydrogenation as described above in Example1(I)(B).

D. Solution Phase Coupling of a `First Unit` and a `Bifunctional Unit`

Aldehyde 3 and hydrazino derivative 8 are coupled in dry CH₂ Cl₂/MeOH/AcOH as described in Example 3 of Ser. No. 08/039,979, filed Mar.30, 1993, to furnish an intermediate hydrazone 9 (L_(S) =CHO). Thelatter product is reduced with NaBH₃ CN/AcOH to furnish a hydrazinolinked molecule 10 (L_(S) =N,N'-diphenylimidazolidino). Subsequently, isbis-alkylated with N1-methylformylthymine to yield 12 (R_(Z) =H, L_(S)=CH₂ OH, R_(Y) =N1-ethylthymine).

The reactive aldehyde moiety of 12 can be regenerated by acid treatmentto deblock the N,N-diphenyl imidazolidine. If compound 5 is used inplace of compound 8, the aldehyde moiety can be regenerated by OsO₄oxidation/NaIO₄ cleavage of the terminal vinyl moiety (i.e., L_(S)=CH═CH₂). Thus, another round of coupling is carried out followed byreduction and alkylation with tether or tether plus a new ligand. Inthis manner, one can place a variety of ligands on a single molecule,separated by an appropriate linear chain, an important feature forrecognition of macromolecules.

The coupling may be terminated at any point by utilizing a terminalunit, such as molecule 11. This compound provides a hydrazino end tocouple with an aldehyde but bears a protected hydroxyl group, which willbe deblocked to provide an hydroxyl moiety.

In addition, one may choose to attach a phosphate or phosphonate groupvia terminal hydroxyl group in order to provide higher solubility tooligomeric unit.

II. Automated Solid Support Synthesis of an Oligomeric Molecule LinkedVia Hydrazino Linkages (FIG. 1)

A. Synthesis of a 4-Penten-1-ol Attached to Solid Support

4-Penten-1-ol is attached via a succinyl linker onto CPG followingstandard protocol (e.g., R. T. Pon in Protocols For Oligonucleotides AndAnalogs, Chapter 24, Agrawal, S., ed., Humana Press, Totowa, N.J.,1993.). The CPG bound 4-penten-1-ol 2 (R_(Z) =CPG, r=1) is oxidized withOsO₄, and the product treated with NaIO₄ to yield 3 with a free aldehydogroup. Next, a reductive coupling with bifunctional unit such as 5furnishes 10 bound on CPG. Subsequent alkylation with a tether such aschloroethane furnishes 12. In a similar manner, the deblocking ofimidazolide with acid and repeated coupling with another bifunctionalunit allows the linear growth of the hydrazino linked oligomer, until adesired length of the molecule is obtained.

The foregoing solid support synthesis can be transferred to a robotic orautomated synthesis technology as, for example, in the generation andrapid screening of libraries of molecules (see, e.g., Zuckermann, et.al., J. Am. Chem. Soc. 1992, 116, 10646).

EXAMPLE 2 Reductive Coupling

Solution Phase Synthesis of Oligomeric Molecule Linked Via AminoLinkages (FIG. 2)

The `first unit` for this synthesis is the same as used in Example 1,above.

A. Synthesis of Bifunctional Units, 4-Pentenyl-1-amine, 15, and 3-(N,N¹-diphenyl imidazolidine)-butyl-1-amine, 13.

Treatment of 4-penten-1-ol 1 (r=1) with methlysulfonyl chloride inpyridine at 0° C. affords the sulfonate, which on treatment with lithiumazide in DMF gives azido derivative 14. Reduction of 14 with tributyltinhydride in dimethyl acetamide furnishes title compound 15.

Yet another bifunctional unit 13 is prepared in five steps, startingfrom 1. The hydroxyl group initially is protected with t-butyldiphenylsilyl group and the product, on oxidative cleavage using OsO₄ /NaIO₄,gives aldehyde 3 (R_(Z) =TBDPS). The latter compound is furthertransformed to the imidazolidine derivative 4, which on desilylationfollowed by conversion of the hydroxyl group to an amino group via anazide, furnishes 13 (see, e.g., Lin, et. al., J. Med. Chem. 1978, 21,109).

B. Coupling of First and Bifunctional Units

To a stirred solution of aldehyde 3, amine 13 and acetic acid in CH₂ Cl₂is added NaBH(OAc)₃ under argon. Alternatively, amine 15 is used inplace of amine 13. The suspension is stirred for 3 h and the reactionmixture, on work up as described in Example 17 of Ser. No. 08/039,979,filed Mar. 30, 1993, gives the dimeric 17 (L_(S) =CHO or CH═CH₂).Reductive amination is performed thereon generally in accordance withTet. Lett., 1990, 31, 5595. Subsequently, the amino functionality isreductively alkylated with N1-methylformylthymine to provide 19 (R_(Z)=H, L_(S) =CH₂ OH, R_(Y) =N1-ethylthymine). Coupling can be repeated toobtain compounds of formula 19 with varying length (e.g. r=1-20).

EXAMPLE 3 Nucleophilic Coupling

Oligomeric Molecules Linked Via Amino Linkages (FIG. 3)

FIG. 3 describes a general method for assembly of amino linked linearmolecules. Further methods are described by Niitsu, et. al., Chem.Pharm. Bull. 1986, 31, 1032.

A. Synthesis of First Unit, 23

The title compound is prepared from commercial 1, 3-propanediol, whichon monosilylation with t-butyldiphenylsilylchloride gives protected 20(R_(Z) =TBDPS, r=1). The free hydroxyl group of 20 is then convertedinto a tosyl leaving group as described by J. March in Advanced OrganicChemistry, Reactions, Mechanisms, and Structure, page 352, John Wiley &Sons, New York, 1992 to furnish 23 (R_(L) =O-tosyl). Other suitableleaving groups include brosylates, nosylates, mesylates or halides.

B. Synthesis of Bifunctional Unit, 22

Treatment of 3-bromo-1-propanol with lithium azide in DMF furnishes3-azido-1-propanol, which on silylation provides 21 (L_(S) =TBDPS, r=1).The azido group of 21 is reduced to provide the bifunctional unit 22.The nitrogen nucleophile at the reactive end of compound 22 is blockedwith a 9-fluorenylmethoxycarbonyl (FMOC) group, and the hydroxyl groupat the dormant end is deblocked and transformed into a reactive ester asin Example 3(A), above to provide 23 (L_(S) =tosyl).

C. Coupling of First Unit and Bifunctional Unit

Compounds 22 and 23 are reacted in presence of an appropriate base tofurnish a secondary amine 24 as the product. Subsequently, amino groupof 24 is reductively alkylated with N1-methylformylthymine to yieldcompound 19 (R_(Z) =H, L_(S) =H, R_(Y) =N1-ethylthymine). In order tocontinue with coupling, the blocking group from hydroxyl moiety isremoved and the resulting hydroxyl group connected to an active estermoiety. Another round of coupling takes place, followed byalkylation/deblocking/esterification steps until a molecule of desiredlength is obtained.

EXAMPLE 4 Reductive Coupling

Solution Phase Synthesis of an Oligomeric Molecule Linked ViaHydroxylamine Linkages (FIG. 4)

A. Synthesis of a `First Unit`, Amino-O-benzylalcohol, 27

Title compound 27 is prepared in two steps starting from commercialbenzyl alcohol 25 (L_(S) =phenyl). In the first step, Mitsunobu reactionof 25 withN-hydroxyphthalimide/triphenylphosphine/diethylazodicarboxylate gives ano-phthalimido derivative 26. Treatment of 26 with methylhydrazine gives27.

B. Synthesis of a `Bifunctional Unit`, 3

Title compound 3 is prepared in a manner described in Example 1, above.

C. Coupling of a `First Unit` and a `Bifunctional Unit`

A mixture of 27, 3, and acetic acid is stirred in CH₂ Cl₂ for 1 h atroom temperature. The solvent is evaporated to furnish the crude oxime28, which on reduction with NaBH₃ CN/AcOH (as described in Example 11 ofSer. No. 08/039,979, filed Mar. 30, 1993) furnishes 29. The amino groupof 29 is further reductively alkylated with N1-methylformylthymine toyield 30 (R_(Z) =H, L_(S) =phenyl, R_(Y) =N1-ethylthymine).

Alternatively, the terminal phthalimido group of 28 is deblocked with 3%methylhydrazine in CH₂ Cl₂ and the o-amino group is coupled with anotherbifunctional unit under acidic conditions. This cycle of treatment canbe repeated with methylhydrazine and coupling until an oligomer ofdesired length is formed. All oxime linkages can be reduced in one stepusing NaBH₃ CN/AcOH treatment, as described above. A common tether or atether and ligand then can be attached in a single alkylation step toyield 30. However, this methodology provides a means to obtain anoligomeric unit with similar tether or tether and ligand placed ontoamino group.

In another method, the oxime linkage is reduced immediately aftercoupling and attachment of the tether or tether and ligand is effected.This modification in the procedure allows placement a tether or tetherand ligand of choice at a preselected position within an oligomer.

EXAMPLE 5 Radical Coupling

I. Solution Phase Radical Coupling Methodology for Linear HydroxylaminoLinked Oligomers (FIG. 5)

A. Synthesis of a `First Unit`, O-Benzylformaldoxime, 31 (L_(S) =phenyl)

The title compound is prepared from benzyl alcohol following a proceduregenerally in accordance with Hart, et. al., J. Am. Chem. Soc. 1988, 110,1631.

B. Synthesis of Bifunctional Unit, 2-Iodo-1-O-phthalimidoethanol, 33

Ethyleneglycol is selectively protected with t-butyldiphenylsilyl groupgenerally in accordance with Nair, et. al., Org. Prep. Procedures Int.1990, 22, 57. A Mitsunobu reaction of the monosilylated ethyleneglycolwith N-hydroxyphthalimide in a manner described by Debart, et. al., Tet.Lett. 1992, 33, 2645, furnishes2-O-tert-butyldiphenylsilyl-1-O-phthalimidoethanol. Deblocking of thesilyl group of this compound with Bu₄ NF/THF, followed by iodinationprovides the desired bifunctional molecule 33.

C. Coupling of a `First Unit` and a `Bifunctional Unit`

Bis(trimethyltstannyl)benzopinacolate mediated intermolecularfree-radical carbon-carbon bond-forming reaction is carried out inbenzene generally in accordance with Example 85 of Ser. No. 08/039,979,filed Mar. 30, 1993, with 31 as a radical acceptor and 33 as a radicalprecursor to yield a linear hydroxylamine 29 (R_(Z) =Phth.).

The amino group of hydroxylamine 29 is reductively alkylated withN1-methylformylthymine to yield 30 (R_(Z) Phth., R_(Y)=N1-ethylthymine). Treatment of 30 with 3% methylhydrazine/CH₂ Cl₂provides a terminal O-amino group, which on formylation with 1 molequivalent of HCHO/MeOH provides an oxime functionality at the reactiveend of 30 (R_(Z) =N═CH₂) for the next round of coupling. Thus, the chainlength is extended by reacting 30 with 33 in a similar manner, followedby alkylation, hydrazinolysis and formylation to obtain the desiredlength of the oligomer. The final, terminal unit 32 is employed when nomore chain elongation is required. Deblocking with Bu₄ NF will furnish aterminal hydroxyl group in oligomeric 30.

II. Solid Support Synthesis

As described in Example 1(II)(A), above, oligomeric molecules areprepared by attaching 31 (L_(S) =CH₂ OH) to a solid support such as CPGor polystyrene via an appropriate linker. Once the oligomer of desiredlength is obtained, the product is cleaved from the support to furnishfully deblocked product, 30.

EXAMPLE 6 Reductive Coupling

Solid Support Synthesis of Covalently Linked Duplex Structures asHairpins/Stem-Loops and Cyclic Oligomeric Structures Via HydroxylaminoLinkages (FIG. 6)

A. Cyclic Oligomers

An appropriate solid support, such as 35 (Y=phenyl) is prepared fromtrisubstituted benzene following a double Mitsunobu reaction describedin Tet. Lett. 1992, 33, 2645 and loading of the product via succinyllinker (Z) onto a CPG support (see, e.g., R. T. Pon in Protocols ForOligonucleotides And Analogs, Chapter 24, Agrawal, S., ed., HumanaPress, Totowa, N.J., 1993.). The CPG bound material is packed into a 1μM column and attached to an ABI DNA synthesizer 380 B model.Bis-phthalimido groups are deblocked with 3% N-methyl hydrazine/CH₂ Cl₂solution to liberate desired bis-O-amino moiety, 35. Then, bifunctionalreagent 3 (R_(Z) =TBDPS) is employed with 5% AcOH/CH₂ Cl₂ to givebis-oxime 38 (r=1). Deblocking with N-methyl hydrazine and coupling with3 is repeated until an oligomeric bis-oxime of desired length isobtained. The CPG loaded 40 is removed from the synthesizer and treatedwith ACOH/NaCNBH₃ to yield reduced hydroxyl amine 39. Subsequently, allamines are reductively alkylated with N1-methylformylthymine to provide40 (R_(Y) =N1-ethylthymine). The terminal bis-phthalimido groups of 40are deblocked with N-methyl hydrazine and final conjugation withbis-aldehyde 37 provides circularized 41, which can be further reduced,alkylated and removed from CPG to yield appropriate circular oligomers,such as 42.

B. Circular/Dumbbelled Oligomers

The method set forth in Example 6(I)(A), above, can be further modifiedto produce molecules that are constructed as linear strands but that onpartial self-hybridization assume defined secondary structures.

Heterobifunctional solid support 36 (Y=phenyl, Z=succinyl, L_(S)=N,N'-diphenyl imidazolidino) is prepared from trisubstituted benzeneaccording to the procedures of Examples 1(I)(A) and 6(I)A). The supportbears a protected aldehydo group on one end, a succinyl linker attachedto the CPG support on a second end, and an O-amino functionality on athird end. Coupling of 3 with 36 provides oxime 43. The product 43 isreduced with NaCNBH₃ /EtOH solution, followed by alkylation withN1-methylformylthymine to provide a ligand 40 (R_(Y) =N1-ethylthymine)with hydrogen bonding capacity. Similarly, deblocking with N-methylhydrazine, followed by coupling with 3, and reductive alkylationprovides a linear sequence bearing nucleic acid bases (A,C,G,T) in adefined order. Elongation of this oligomer is terminated when anappropriate length is achieved. The oligomer is detached from the CPGand purified by HPLC. The pure oligomer is able to self-hybridize toprovide either circular or dumbbell structures of any length.

C. Hairpin/Stem-Loop Duplexes

In order to prepare partially or fully self-complementary molecules,synthesis is commenced with a molecule bearing two functionalities. Oneof these functionalities is the reactive end of the molecule and theother remains dormant/protected. Therefore, a heterobifunctionalmolecule is attached to the CPG to give protected 36, which is deblockedwith N-methyl hydrazine to yield 36 with a free O-amino group. As inExample 6(I)(B), above, coupling with 3 in presence of acetic acidprovides oxime 43. In two steps, the oxime is reduced and alkylated withan appropriate nucleic acid base (such as A,C,G,T) via a tether tofurnish 44. The chain is elongated utilizing a three step process(deblocking, then coupling, then reductive alkylation) until an oligomerof desired length is obtained. Finally, the linear molecule is deblockedfrom CPG and dissolved in salt-buffer to provide a self complementarysecondary structure as per the preorganized nucleic acid bases.

The protected end of the molecule is deblocked and utilized for asite-specific cross-linking on the complementary strand. Suchcross-linked molecules are expected to provide additional conformationaland structural stability to maintain a duplex hairpin or stem-loop ordumbbelled shape.

EXAMPLE 7 Solid Support Synthesis of Covalently LinkedDuplex/Hairpins/Stem-Loops and Cyclic Oligomers Via Amino Linkage

FIG. 7 describes one method for assembly of amino linked duplexes orcircular oligomers. Tuladhar, et. al., Tet. Lett. 1992, 33, 2203,describes a synthetic route for the preparation of poly-N-N¹-dimethylethylenediamines, which method can be adapted for preparationof the title oligomers.

A. Circular Polyamine, 55

A bis-N-alkylated phenyl amine bearing a tether, T, and a ligand, L, isconjugated to CPG via standard procedures (see, e.g., R. T. Pon inProtocols For Oligonucleotides And Analogs, Chapter 24, Agrawal, S.,ed., Humana Press, Totowa, N.J., 1993.) to provide 48 (Y=phenyl,Z=succinyl, R_(Y) =N1-alkylated pyrimidine bases or N9-alkylated purinebases). A complete set of appropriately alkylated amine building blocks50 (R_(L) =O-tosyl, R_(Y) =N1-ethylthymine, R_(Z) =FMOC) next areprepared with a leaving group and protected secondary amine at oppositeends. Nucleophilic displacement of the leaving group of 50 bybis-N-alkylated 48 in presence of an appropriate base, such as K₂ CO₃ ortriethylamine results in formation of branched 51. The protecting groupof the bis-amino function is removed and yet another round of basecatalyzed coupling furnishes a longer oligomer. Thus, repetition ofdeblocking and coupling provides a molecule of desired length. To closethe loop or tie the two amino branched, compound 53 having bis-leavinggroups (R_(L)) are employed to provide a circularized oligomer 54. Theoligomer is then deblocked from the support in the standard manner as isutilized in solid phase oligonucleotide synthesis (see, e.g.,Oligonucleotide Synthesis, Gait, M. J., ed., IRL Press, Oxford, 1984.)

Alternatively, 51 is deblocked after a desired length is achieved toprovide a linear oligomer. This oligomer is circularized bytemplate-directed coupling, wherein a short complementary oligomer isemployed to hybridize the loose ends and then carry out the couplingwith 53 to provide compound 55. Kool, et. al., J. Chem. Soc. Chem.Commun. 1991, 1161, have reported similar ligation of reactive ends(utilizing a template) to yield circularized products.

B. Hairpin and Stem-Loops Linked Via Polyamines

As described in Example 6(I)(C), above, self-complementary hairpin andstem-loop structures are prepared in accordance with FIG. 7. Synthesisis accomplished by alkylation of N-alkyl amine 50 (R_(L) =I) withmonoamine 49 (L_(S) =N,N'-diphenyl imidazolidino) to furnish 52. Use ofan iodo leaving group in 50 is preferred, due to high couplingefficiency. Also preferred is use of a bifunctional reagent 50 whichalready bears a functional group residue attached via a tether. Thus, itis possible to incorporate appropriate ligands, e.g. heterocyclic basesor substituted aryl groups, one at a time to introduce the desiredrecognition element into the growing oligomer. Once an oligomer ofexpected length is obtained, it is removed from the support by standardmethods.

The oligomer is allowed to anneal under appropriate salt concentrationsto provide a hairpin or stem-loop structure. The development of thesemethods for cationic polyamine synthesis are attractive because theirunique interaction with anionic biological target molecules and presenceof an active uptake system in a variety of cell types.

EXAMPLE 8 General Procedure for Linear Tertiary Nitrogen CombinatorialLibraries and SURF Deconvolution

Description of general process

This example describes the general procedure for creation anddeconvolution of a library of tertiary nitrogen based oligomericcompounds. Specific synthetic details corresponding to solution phaseand solid support phase synthesis utilizing the procedure of thisgeneral description, alternate extenders and extender synthesis are setforth in companion Examples 9 to 18 below.

In reference to FIGS. 8 to 13 and to the companion examples, illustratedis the synthesis of a 1,4,9,14,19-pentaaza-8,12,18-trioxanonadecane withnitrogens 1,4,9,14,19 combinatorialized with four benzyl moieties asletters. A total of 1024 compounds are prepared in four sets of 256compounds each. The procedures describe the preparation of the four setsof linear polyamine/oxyamines compounds 122a-d (round 1, FIG. 9). Eachset has positions 1 through 5 (positions 1 and 5 are primary nitrogenswhere as positions 2, 3 and 4 are secondary oxyamine nitrogens)substituted in a combinatorial manner, i.e. combinatorialized, withequal amounts of the letters. For illustrative purposes, benzyl,m-methylbenzyl, m-nitrobenzyl, and m-methoxybenzyl moieties are theselected letters. The precursors compounds for the letters all arecommercially available compounds purchased from Aldrich Chemicals,Milwaukee, Wis. At the completion of synthesis of the library, each sethas position 5 (a primary oxyamine nitrogen) substituted exclusivelywith a known one of the four letters. In the initial approach, theletter selected to be fixed is placed in the molecule last. Forillustrative purposes position 5 was selected as the fixed position inthe first round synthesis of the libraries (FIG. 9). The iterativedeconvolution process (SURF) requires four subsequent rounds ofsynthesis (FIGS. 10-13) to be performed to identify the most activemolecules. Each round of synthesis is performed to allow the positionselected to be fixed as the last fixed position. Other positionselection approaches can be taken, e.g. the first selected position isfixed. To fix a position last in rounds 3-6, the acid labile sulfenyltriphenyl methyl moiety is utilized to protect the designated nitrogenatom until combinatorialization of other positions and fixing of knownpositions is completed.

The linear polyamine/oxyamines are prepared by two sets of sequentialreactions: submonomer addition of a letter to a secondary nitrogen andextension (elongation) of the chain to provide another secondarynitrogen for combinatorializtion via submonomer chemistry (first step).The purpose of repeating these sequential sets of reactions is toliberate/provide a reactive secondary amine (the next position to becombinatorialized) in the growing chain in the absence of other reactivecenters and to extend the molecular length (and thus the number ofcombinatorial positions) in the polyamine/oxyamine chain.

The key starting material for this particular combinatorial chemistrylibrary is 1-(tert-butoxycarbonyl)-9-phthaloyl-1,4,9-triaza-8-oxa-decane[t-Boc-NH--(CH₂)₂ --NH--(CH₂)₃ --O--N-phthaloyl, 106, where BG=t-Boc inthe figures. This synthesis is depicted in FIG. 8. Solid phase synthesisof combinatorial libraries is effected in the same manner as depicted inFIG. 8 using compounds set forth in Examples 11 and 12. In this instancethe same key intermediate starting compounds are used for effecting thecombinatorial synthesis on a solid support where BG=solid support, e.g.1-(BG)-9-phthaloyl-1,4,9-triaza-8-oxa-decane [BG-NH--(CH₂)₂ --NH--(CH₂)₃--O--N-phthaloyl, 109].

For the solution phase synthesis, the compound 106 is protected at oneend (position 1) with the acid labile tert-butoxycarbonyl group (t-Boc)and the terminal oxyamine (position 3) is protected by a base labilephthaloyl group (acid dephtholyation can also be used if desired). Theinternal secondary oxyamine (position 2) is unprotected and availablefor submonomer chemistry as shown in compound 106. In addition, compound106 with its internal secondary amine protected with asulfenyltriphenyl, compound 133, is employed in preparations of latterrounds of deconvolution.

    t-Boc--NH--(CH.sub.2).sub.2 --NH--(CH.sub.2).sub.3 O--N-phthaloyl(106)

    t-Boc--NH--(CH.sub.2).sub.2 --NSCPh.sub.3 --(CH.sub.2).sub.3 O--N-phthaloyl(133)

In preparing libraries of compounds, advantage is taken of having threedifferent tertiary nitrogens (a primary amine and a primary oxyamine anda secondary oxyamine) and using protecting groups to alternately protector expose these nitrogen atoms such that they can be functionalized withvarious letters. The selected protecting groups are tert-butoxycarbonyl(t-Boc) and the sulfenyltriphenyl [(S(Ph)₃ ] moieties that are remove byvarious differential acid conditions and the phthaloyl moiety that istypically removed with hydrazines (basic conditions) and with certainacid conditions. For addition of letters, the monomer and the submonomerapproaches are utilized. The submonomer approach requires the additionof a letter intermediate to one of the tertiary nitrogens. This isaccomplished by several chemistries including "Schiff's base reductivealkylation," alkylation, e.g., with alkyl halides, and amide bondformation with acid, acid halides, esters, etc. The Schiff's basereductive alkylation is described in Examples 9 and 10. Otherchemistries is described in Examples 14 and 15. Moieties required forsubmonomer letter addition are aldehydes, ketones, primary and secondaryalkyl halides, sulfonates, trifilates, diazonium salts, acids, acidhalides, esters, etc. These same reactive moieties are employed toextend the chain when attached to an alkyloxyphalimide or a substitutedalkyloxyphalimide (both in monomer and submonomer approaches). Startingmaterials for these reaction are commercial chemical reagents availablefrom various commercial chemical supply houses. They are used aspurchased without further modification.

First Set of Reactions

Submonomer chemistry combinatorilizes position 2 with four aromaticaldehydes, letters L₁₋₄. For this example they are selected asbenzaldehyde, L₁ ; m-tolualdehyde, L₂ ; m-anisaldehyde, L₃ ; and3-nitrobenzaldehyde, L₄, by a split solution procedure. This providesthe aldehydes as benzyl derivatives after reduction of the iminiumintermediates with NaCNHB₃ (via reductive alkylation). Each letter isreacted separately with 106. After purification, if needed, an equalmolar amount of each pure "benzylated" 106 is mixed together to providea mixture of four compounds, 114a-d, with position 2 combinatorializedwith four letters to give compounds 115.

    t-Boc--NH--(CH.sub.2).sub.2 --NL.sub.1-4 --(CH.sub.2).sub.3 O--N-phthaloyl(115)

Second Set of Reactions

Position 3 is deprotected (dephthaloylation with methylhydrazine),followed by reductive alkylation (Schiff's base formation and reductionof imino intermediate with NaCNBH₃) with a selected extender. In thiscase, the selected extender is N-(3-hydroxypropionaldehyde)phthalimide,105.

    OHC(CH.sub.2).sub.2 O-NPhth                                (105)

A variety of other extenders can be employed including aldehydes,ketones, halides, acid halides and the like.

The reaction with the extender provides a reactive secondary nitrogenready for combinatorialization.

    t-Boc--NH--(CH.sub.2).sub.2 --NL.sub.1-4 --(CH.sub.2).sub.3 O--NH--(CH.sub.2).sub.3 --NPhth                           (117)

The sequential set of reaction are repeated again. This is continueduntil the desired length is obtained, i.e. the number of combinatorialsites, the length of molecule, the molecular weight, etc.). In thepresent example, compound 117 (having four sites forcombinatorialization) is extended one additional time to provide fivesite for combinatorialization. Then position 1 is liberated with acidconditions. The resulting primary amine is treated separately with eachof the four aromatic aldehydes. In this case, reduction of theintermediate imino (--CH═N--) moiety is not performed until the finalstep to allow a cleaner reactions when position 5 is subsequently fixedwith each specific letter. These set of reactions providepolyamine/oxyamine compounds 121.

    L.sub.1-4 CH═N--(CH.sub.2).sub.2 --NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O-NPhth(121)

The final set of reactions for the preparation of this particularlibrary provides a fixed letter at position 5. In this case position 5is not combinatorialized--it is not a mixture of the four letters. Eachset of the library has a known letter at position 5. The final reductionwith NaCNBH₃ converts both positions 1 and 5 into benzyl moieties. Thusfour subsets of libraries, compounds 122a-d, (mixtures of compounds) areobtained.

    L.sub.1-4 NH--(CH.sub.2).sub.2 --NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O--NHCH.sub.2 PhH                                         (122a)

    L.sub.1-4 NH--(CH.sub.2).sub.2 --NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O--NHCH.sub.2 PhMe                                        (122b)

    L.sub.1-4 NH--(CH.sub.2).sub.2 --NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O--NHCH.sub.2 PhOMe                                       (122c)

    L.sub.1-4 NH--(CH.sub.2).sub.2 --NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O--NL.sub.1-4 --(CH.sub.2).sub.3 O--NHCH.sub.2 PhNO.sub.2                                  (122d)

This approach employed a split solution process utilizing SURFdeconvolution (iterative screening). Purification by simple flash silicagel column chromatography is performed at any stage as needed.

EXAMPLE 9 Synthesis of Intermediates for Solution and Solid PhaseCombinatorial Synthesis

tert-Butyl N-(2-bromoethyl) Carbamate (101)

Triethyl amine (11 mL, 77 mmole) and di-tert-butyl dicarbamate (15.2 mL,66.5 mmole) were added to 2-bromoethyl-amine hydrobromide (14.3 g, 70mmole) in CH₃ CN (250 mL). The reaction mixture was stirred at roomtemperature for 12 hours under an argon atmosphere. 200 mL of saturatedNaHCO₃ (aq) was added and the stirring was continued for 15 minutes. Themixture was extracted several times with ether, the combined etherlayers were dried over Na₂ SO₄, the Na₂ SO₄ was filtered and thefiltrate was evaporated to give 15.28 g (97.4%) of the title compound:TLC (Rf: 0.7; 10% MeOH/CH₂ Cl₂), ¹ H NMR (CDCl₃) δ 1.5 (s, 9 H,tert-butyl CH3), 3.5 (m, 4 H, CH2), 5.1 (s, 1H, NH). ¹³ C NMR (CDCl₃): δ28.3 (CH3), 32.7 (CH2), 42.3 (CH2), 79.7 (C(CH3)3), 155.5 (CO).

tert-Butyl N-(2-azidoethyl) Carbamate (102)

Sodium azide (5.0 g, 75 mmole) was added to compound 101 (15.28 g, 68.2mmole) in DMF (200 mL). The reaction mixture was stirred at about 80° C.for 12 hours under an argon atmosphere. The reaction mixture was cooledand diluted with 400 mL of ether. The ether layer was washed five timeswith saturated NaCl and dried over Na₂ SO₄. The Na₂ SO₄ was filtered andthe filtrate was evaporated to give 9.8 g (77.1%) of the title compound:TLC (Rf: 0.4; 20% EtOAc/Hexane), ¹ H NMR (CDCl₃) δ 1.4 (s, 9 H,tert-butyl CH3), 3.2 (t, 2H, CH2), 3.3 (m, 2 H, CH2), 4.9 (s, 1 H, NH).¹³ C NMR (CDCl₃): δ 28.2 (CH3), 40 (CH2), 51.1 (CH2), 79.7 (C(CH3)3),155.7 (CO).

tert-Butyl N-(2-aminoethyl) Carbamate (103)

Triphenyl phosphine (15 g, 58 mmole) was added to compound 102 (9.8 g,52.6 mmole) in THF (200 mL) and H₂ O (0.8 mL). The reaction mixture wasstirred at about 80° C. for 12 hours under an argon atmosphere. Thereaction mixture was evaporated to obtain a white solid residue. 200 mLof 0.5 M NaH₂ PO₄ was added, the mixture was stirred and extracted withEtOAc. The aqueous layer was added to 3 N NaOH and extracted with ether.The ether layer was dried over Na₂ SO₄. The Na₂ SO₄ was then filteredand the filtrate was evaporated to give 8.1 g (96.5%) of the titlecompound: TLC (Rf: 0.2; 20% MeOH/CH₂ Cl₂), ¹ H NMR (CDCl₃) δ 1.3 (s, 2H, NH2), 1.4 (s, 9 H, tert-butyl CH3), 2.8 (t, 2 H, CH2), 3.2 (m, 2 H,CH2), 4.8 (s, 1 H, NH). ¹³ C NMR (CDCl₃): δ 28.4 (CH3), 41.9 (CH2), 43.5(CH2), 79.2 (C(CH3)3), 156.2 (CO).

N-(3-Hydroxypropionaldehyde Dimethylacetal)phthalimide (104)

A mixture of 3-bromopropionaldehyde dimethyl acetal (Aldrich Chemical),N-hydroxyphthalimide (Aldrich Chemical), triethylamine, and DMF isheated at 60° C. for five hours and evaporated to dryness under reducedpressure. The residue is distributed between water and ethyl acetate.The organic layer is removed, dried (MgSO₄), and evaporated to drynessunder reduced pressure. The residue is purified by flash, silica gelchromatography to provide 104.

N-(3-Hydroxypropionaldehyde)phthalimide (105)

A mixture of N-(3-hydroxypropionaldehyde dimethylacetal)phthalimide(104) in HCl/KCl buffer (pH 1, 10/30) is stirred at 20-60° C. for 5-24hours and then evaporated to dryness under reduced pressure. The residueis purified by flash, silica gel chromatography to provide 105.

1-(tert-Butoxycarbonyl)-9-phthaloyl-1,4,9-triaza-8-oxa-nonane(t-butoxycarbonyl--NH--(CH₂)₂ --NH--(CH₂)₃ --O--N-phthaloyl) (106)

A mixture 103 (1.1 equivalent) and 105 (1 equivalent) inacetonitrile/toluene (1:1) containing several drops of glacial aceticacid is heated at 20-80° C. for 5-24 hours and then treated with NaCNBH₃(1.1 equivalents). The reduction is allowed to proceed for 1-5 hours.The mixture is washed with aqueous NaHCO₃. The organic layer isseparated and evaporated to dryness under reduced pressure. The residueis purified by flash, silica gel chromatography to provide 106.

1,4,9-triaza-8-oxa-nonane-9-phthaloyl (NH₂ --(CH₂)₂ --NH--(CH₂)₃--O--N-phthaloyl) (107)

A mixture of 106 and 50% aqueous trifluroactic acid/methylene chloride(1:1) is heated at 20-50° C. for 1-24 hours and treated with saturatedNaHCO₃. The mixture is evaporated to dryness under reduced pressure andthe residue purified by flash silical gel chromatography to provide 107.

1-(p-Toluenesulfonyl)-3-chloro-propanol (103a)

To 3-chloro-1-propanol (5.02 mL, 60 mmole) in CH₃ CN (200 mL) was added,p-toluenesufonyl chloride (17 g, 90 mmole) and pyridine (7.3 mL, 90mmole). The reaction mixture was stirred at room temperature for 12hours under an argon atmosphere. 200 mL of saturated NaHCO₃ (aq) wasadded and the stirring was continued for 15 minutes. The mixture wasextracted several times with CH₂ Cl₂. The combine CH₂ Cl₂ layers weredried over Na₂ SO₄, the Na₂ SO₄ was filtered and the filtrate wasevaporated to give 12.35 g (83.8%) of the title compound: TLC (Rf: 0.5;20% EtOAc/Hexane), ¹ H NMR (CDCl₃) δ 2.1 (m, 2 H, CH2), 2.5 (s, 3 H,CH3), 3.6 (t, 2 H, CH2), 4.2 (t, 2 H, CH2), 7.6 (d & d, 4 H, Ar). ¹³ CNMR (CDCl₃): δ 21.4 (CH3), 31.5 (CH2), 40.2 (CH2), 66.7 (CH2), 127.8(Ar), 129.8 (Ar), 132.5 (Ar), 144.9 (Ar).

1-(Methanesulfonyl)-3-chloro-propanol (103b)

To 3-chloro-1-propanol (5.02 mL, 60 mmole) in CH₃ CN (200 mL) was addedmethanesulfonyl chloride (10.4 g, 90 mmole) and triethyl amine (13 mL,90 mmole). The reaction mixture was stirred at room temperature for 12hours under an argon atmosphere. 200 mL of saturated NaHCO₃ (aq) wasadded and the stirring was continued for 15 minutes. The mixture wasextracted several times with ether. The combined ether layers were driedover Na₂ SO₄, the Na₂ SO₄ was filtered and the filtrate was evaporatedto give 10 g (96.6%) of the title compound: TLC (Rf: 0.3; 20%EtOAc/Hexane), ¹ H NMR (CDCl₃) δ 2.2 (m, 2 H, CH2), 3.0 (s, 3 H, CH3),3.6 (t, 2 H, CH2), 4.4 (t, 2 H, CH2). ¹³ C NMR (CDCl₃): δ 31.5 (CH2),37.0 (CH3), 40.2 (CH2), 66.3 (CH2).

tert-Butyl N¹ -(2-aminoethyl)-N² -(3-chloropropyl)carbamate (110)

To compound 103b (4.4 g, 25 mmole) in THF (40 mL) was added to thecompound 103 (9.8 g, 52.6 mmole). The reaction mixture was added tosodium hydride (0.23 g, 5.7 mmole) in an ice bath. The reaction mixture,under an argon atmosphere, was allowed to warm up to room temperatureand stirring continued for 12 hours. 100 mL of 0.5 M NaH₂ PO₄ (aq) wasadded and the aqueous layer was extracted with toluene. The aqueouslayer was added to 3 N NaOH and extracted with ether. The ether layerwas dried over Na₂ SO₄. The Na₂ SO₄ was filtered and the filtrate wasevaporated to give an oily residue. The residue was purified by flashchromatography over silica gel using MeOH/CH₂ Cl₂ as the eluent. Thepure fractions were pooled together and evaporated to dryness to give0.45 g (49.5%) of the title compound: TLC (Rf: 0.5; 20% MeOH/CH₂ Cl₂), ¹H NMR (CDCl₃) δ 1.4 (s, 9 H, tert-butyl CH3), 1.9 (m, 2 H, CH2), 2.7 (q,4 H, CH2), 3.2 (m, 2 H, CH2), 3.6 (t, 2 H, CH2), 4.9 (s, 1 H, NH). ¹³ CNMR (CDCl3): δ 28.4 (CH3), 32.7 (CH2), 40.3 (CH2), 42.9 (CH2), 46.4(CH2), 49.1 (CH2), 79.3 (C(CH3)3), 156.2 (CO).

tert-Butyl N¹ -(2-aminoethyl)-N² -tritylsulfenyl-N² -(3-chloropropyl)carbamate, [1-(t-butoxycarbonyl)-NH--(CH₂)₂ --NS(Ph)₃ --(CH₂)₃ --Cl](111)

Pyridine (0.7 mL, 8.5 mmole) and triphenyl methanesulfenyl chloride(0.42 g, 1.36 mmole) were added to compound 110 (420 mg, 1.7 mmole) inCH₂ Cl₂ (30 mL). The reaction mixture was stirred for 12 hours under anargon atmosphere. 20 mL of 0.5 M NaH₂ PO₄ (aq) was added and the aqueouslayer was extracted several times with CH₂ Cl₂. The CH₂ Cl₂ layer wasdried over Na₂ SO₄, the Na₂ SO₄ was filtered and the filtrate wasevporated to give an oily residue. The residue was purified by flashchromatography over silica gel using EtOAc/Hexane as the eluent. Thepure fractions were pooled together and evaporated to dryness to give150 mg (17.3%) of the title compound: TLC (Rf: 0.5; 20% EtOAc/Hexane), ¹H NMR (CDCl₃) δ 1.4 (s, 9 H, tert-butyl CH3), 1.9 (m, 2 H, CH2), 2.9 (m,4 H, CH2), 3.2 (m, 2 H, CH2), 3.4 (t, 2 H, CH2), 4.6 (s, 1 H, NH). ¹³ CNMR (CDCl₃): δ 28.4 (CH3), 29.7 (CH2), 37.9 (CH2), 42.2 (CH2), 53.4(CH2), 56.5 (CH2), 79.3 (C(CH3)3), 127.2 (Ar), 127.9 (Ar), 130.1 (Ar),143.0 (Ar), 156.2 (CO).

tert-Butyl N¹ -(2-aminoethyl)-N² -(3-aminooxypropanyl) Carbamate(1,4.9-triaza-8-oxa-1-(t-Boc)-nonane] (112)

Sodium carbonate (80 mg, 7.5 mmole) and N-hydroxyphthalimide (0.12 g,0.75 mmole) were added to compound 110 (420 mg, 1.7 mmole) in DMF (5mL). The reaction mixture was stirred at 80° C. for 6 hours followed bystirring at room temperature for 12 hours under an argon atmosphere. Thereaction mixture was filtered, 20 mL of H₂ O was added and the aqueouslayer was extracted several times with ether. The ether layer was driedover Na₂ SO₄, the Na₂ SO₄ was filtered and the filtrate was evporated togive the title compound: TLC (Rf: 0.4; 20% MeOH/Hexane), ¹ H NMR (CDCl₃)δ 0.9 (m, 1 H, NH), 1.4 (s, 9 H, tert-butyl CH3), 1.8 (s, 2 H, NH2), 2.1(m, 2 H, CH2), 2.5 (t, 2 H, CH2), 3.1 (m, 2 H, CH2), 3.2 (t, 4 H, CH2),4.9 (s, 1 H, NH). ¹³ C NMR (CDCl₃): δ 28.4 (CH3), 29.7 (CH2), 38.4(CH2), 55.2 (CH2), 58.8 (CH2), 75.9 (CH2), 79.3 (C(CH3)3), 156.2 (CO).

1-(tert-Butoxycarbonyl)-9-phthaloyl-4-(triphenylsulfenyl)-1,4,9-triaza-8-oxa-nonane(t-Boc--NH--(CH₂)₂ --N[S(C₆ H₅)₃ ]--(CH₂)₃ --O-Nphthaloyl) (113)

A mixture of chloride compound 111 (100 mmol), dry dimethylformamide(DMF), N-hydroxyphthimide (110 mmol), NaI (10 mmol), and triethyl amine(110 mmol) is heated at 50° C. for 1-24 hours. The mixture is evaporatedto dryness under reducted pressure and the residue is distributedbetween water and ethyl acetate. The organic layer is dried (MgSO₄) andevaporated to dryness to provide protected polyamine 113. The materialis purified by flash silica gel chromatography to give 113.

EXAMPLE 10 Synthesis of First Round Library from Protected Polyamine 109

(BG=t-Boc) and Four Letters (benzaldehyde, [L₁ ]; m-tolualdehyde, [L₂ ];m-anisaldehyde, [L₃ ]; and 3-nitrobenzaldehyde, [L₄ ].

Step A

t-Boc polyamine 109 (BG=t-Boc) (42.1 g, 125 mmol) is divided into fourequal parts and each is reacted separately with benzaldehyde (L₁)(Aldrich, catalog #B133-4), m-tolualdehyde (L₂) (Aldrich, catalog#T3,550-5), m-anisaldehyde (L₃) (Aldrich, catalog #12,965-8), or3-nitrobenzaldehyde (L₄) (Aldrich, catalog #N1,084-5). The reactants aredissolved in an organic solvent selected from methylene chloride,dichloroethane, ethyl acetate, toluene, or methanol, suitable for theindividual reactants. For each reaction, 1.5-3 equivalents of thealdehyde is employed with glacial acetic (1-3%) acid added as acatalyst. The reactions are allowed to proceed from 5-24 hours thentreated directly with NaCNBH₃ (2-3 equivalents). The reduction reactionmixtures are stirred at room temperature for 1-10 hours, filtered andevaporated to dryness under reduced pressure. The residue is suspendedbetween ethyl acetate and aqueous NaHCO₃. The organic layer isseparated, dried (MgSO₄), and concentrated to dryness under reducedpressure. The individual residues may be purified by columnchromatography if needed. The procedure provides a polyamine/oxyamine115 with position 2 combinatorialized with the four selected aromaticaldehydes (reductive alkylation to provide benzyl moieties).

Steps B & C

Equal moles of each pure reaction residue (114a-b, ≈11.0 g, ≈25 mmoleach) are dissolved in methanol and mixed together. The solution istreated with methylhydrazine (250 mmol), heated under reflux for onehour, and evaporated under reduce pressure. The residual mixture istriturated with chloroform and filtered. The filtrate is evaporated todryness under reduced pressure and the residue purified by silica gel,flash column chromatography if needed to provide ≈100 mmol (≈31 g) ofoxyamine 116.

Step D

Oxyamine mixture 116 (≈31 g, ≈100 mmol) dissolved in ethyl acetatecontaining glacial acetic acid (1-3%) is treated withN-(3-hydroxypropionaldehyde)phthalimide (105, 110 mmol). The solution isstirred at room temperature for 1-24 hours followed by treating withNaCNBH₃ (150 mmol) and stirring at room temperature for 1-10 hours. Thereaction mixture is poured into H₂ O and the layers separated. Theorganic phase is washed with saturated NaHCO₃ solution, dried (MgSO₄),and evaporated under reduced pressure. The residual 117 is purified byflash silica gel column chromatography if needed to yield approximately100 mmol (≈50 g) of 117.

Step E (Repeat Step A & B), Combinatorilization of Position 3 to ProvidePolyamine/Oxyamine 118

The t-Boc protected polyamine 117 (50.6 g, 100 mmol) is divided intofour equal parts and each is reacted separately with benzaldehyde (L₁),m-tolualdehyde (L₂), m-anisaldehyde (L₃), or 3-nitrobenzaldehyde (L₄).The reactants are dissolved in an organic solvent selected frommethylene chloride, dichloroethane, ethyl acetate, toluene, or methanol,suitable for the individual reactant. For each reaction, 1.5 to 3equivalents of the aldehyde is used with glacial acetic acid (1-5%)employeed as a catalyst. The reactions are allowed to proceed from 5-24hours then treated directly with NaCNBH₃ (2-3 equivalents). The reactionmixtures are stirred at room temperature for 1-10 hours, filtered andevaporated to dryness under reduced pressure. The residue is suspendedbetween ethyl acetate and aqueous NaHCO₃. The organic layers areseparated, dried (MgSO₄), and concentrated under reduced pressure. Thefour individual reactions may be purified by column chromatography ifneeded. Equal mole equivalents of each pure reaction residue isdissolved in methanol and mixed together to provide 118 (≈60 g)

Steps F & G, (repeat of Steps C, D & E), Combinatorilization of Position4 to Provide Polyamine/Oxyamine 120

Step F

A solution of 118 (60.7 g, 100 mmol) in methanol is treated withmethylhydrazine (250 mmol), heated under reflux for one hour, andevaporated under reduce pressure. The residual mixture is trituratedwith chloroform and filtered. The filtrate is evaporated to drynessunder reduced pressure and the residue purified by silica gel, flashcolumn chromatography if needed to provide ≈100 mmol (≈75 g) of position5 oxyamine, 119. The oxyamine compound is dissolved in ethyl acetatecontaining glacial acetic acid (1-3%) and treated withN-(3-hydroxypropionaldehyde)phthalimide (105, 110 mmol). The solution isstirred at room temperature for 1-24 hours followed by treating withNaCNBH₃ (150 mmol) and stirring at room temperature for 1-10 hours. Thereaction mixture is mixed with water and the layers separated. Theorganic phase is washed with saturated NaHCO₃ solution, dried (MgSO₄),and concentrated under reduced pressure. The residual 119 is purified byflash silica gel column chromatography if needed to yield approximately100 mmol (≈75 g) of 119.

Step G

The t-Boc polyamine 119 (≈75 g, ≈100 mmol) is divided into four equalparts and each is reacted separately with benzaldehyde (L₁),m-tolualdehyde (L₂), m-anisaldehyde (L₃), and 3-nitrobenzaldehyde (L₄)as described in Step E above. The resulting four individual reactionsare purified by column chromatography as needed. Equal mole equivalentsof each pure reaction residue is dissolved in methanol and mixedtogether to provide 120 (≈89 g).

Step H Combinatorilization of Position 1 to Provide Polyamine/Oxyamine121

Residue 120 (90 g, 100 mmol) dissolved in a mixture of 1:1 volume of 50%aqueous trifluoroacetic acid and dichloromethane is reacted at 20-50° C.for 1-24 hours and treated with NaHCO₃ solution. The organic layer isseparated, dried (MgSO₄) and evaporated to dryness under reducedpressure. The residue may be purified by chromatography as needed. Theresidue (≈100 mmol) is dissolved in methanol and divided into four equalparts and each evaporated under reduced pressure to dryness. Eachresidue is reacted separately with benzaldehyde (L₁), m-tolualdehyde(L₂), m-anisaldehyde (L₃), or 3-nitrobenzaldehyde (L₄) as describedabove. The reactions are not treated with NaCNBH₃ in order to allowisolation of the imino derivative of each aromatic aldehyde. The fourindividual reactions are purified by column chromatography as needed.Equal mole equivalents of each pure reaction residue is dissolved inmethanol and mixed together to provide 121 (≈86 g).

Step I Combinatorilization of Position 5 to Provide Polyamine/Oxyamine122a-d as Four Sets of First Round Libraries with Position 5 Fixed

Mixture 121 (≈86 g, ≈100 mmol) in methanol is treated withmethylhydrazine (150 mmol) at 20-50° C. for 1-24 hours and thenevaporated under reduce pressure. The residual mixture is trituratedwith chloroform and filtered. The filtrate is evaporated to drynessunder reduced pressure and the residue purified by silica gel flashcolumn chromatography as needed to provide ≈100 mmol of position 5oxyamine 121. The oxyamine 121 is dissolved in ethyl acetate andseparated into four equal parts. Each solution is reacted separatelywith benzaldehyde (L₁), m-tolualdehyde (L₂), m-anisaldehyde (L₃), or3-nitrobenzaldehyde (L₄) as described above. The reactions are allowedto proceed from 5-24 hours, then treated directly with NaCNBH₃ (2-3equivalents). The reaction mixtures are stirred at room temperature for1-10 hours, filtered and evaporated to dryness under reduced pressure.The residues are suspended between ethyl acetate and aqueous NaHCO₃. Theorganic layers are separated, dried (MgSO₄), and concentrated to drynessunder reduced pressure. The four individual reactions are purified bycolumn chromatography as needed to give a library of four sets ofpolyamine/oxyamines combinatorialized at positions 1-4 with four benzylmoieties and fixed at position 5 with a single, known benzyl moiety.

EXAMPLE 11 Formation of Activated Derivative for Attachment to CPG SolidPhase Support

I. General Procedure Amino Compounds

I-a Ethyl Linker--O-Succinyl Ethyl N-phthalimide

Succinic anhydride (1.5 g, 15 mmole) and DMAP (1.84 g, 15 mmole) wereadded to 2-hydroxyl-ethyl N-phthalimide (1.9 g, 10 mmole) dissolved in40 mL of CH₂ Cl₂ (40 mL). The reaction mixture was stirred at roomtemperature for 12 hours under an argon atmosphere. NaH₂ PO₄ (10% aq,100 mL) was added and the stirring was continued for 15 minutes. Theaqueous layer was extracted several times with CH₂ Cl₂. The combined CH₂Cl₂ was dried over Na₂ SO₄ and the filtrate concentrated to give ayellow residue. The residue was purified by flash chromatography oversilica gel using Hexane/EtOAc as the eluent. The pure fractions werepooled together and evaporated to dryness to give 2.31 g (79.3%) of thetitle compound: TLC (Rf: 0.5; 80% EtOAc/Hexane), ¹ H NMR (DMSO-d6) δ 2.4(s, 4 H, succinyl CH2), 3.8 (t, 2 H, CH2), 4.2 (t, 2 H, CH2), 7.8 (d, 4H, Ar), 12.2 (s, 1 H, COOH).

II. General Procedure Hydroxylamino Compounds

II-a Ethyl Linker--O-Succinyl Ethyl Hydroxy N-phthalimide

Diethylazodicarbonate (3.15 mL, 20 mmole) in THF (8 mL) was added to aTHF solution of N-hydroxy phthalimide (3.26 g, 20 mmole), ethyleneglycol (1.2 mL, 20 mmole) and triphenyl phosphine (5.25 g, 20 mmole).The reaction mixture was stirred at room temperature for 12 hours underan argon atmosphere. The reaction mixture was evaporated to dryness andtriturated with ethyl ether. The mixture was filtered to remove a whiteppt and the filtrate was concentrated to a residue. The residue was usedfor the next step without further purification. TLC (Rf: 0.7; 5%MeOH/CH₂ Cl₂), ¹ H NMR (DMSO-d6) δ 3.7 (q, 2 H, CH2), 4.2 (t, 2 H, CH2),7.9 (s, 4 H, Ar). ¹³ C NMR (DMSO-d6): δ 59.2 (CH2), 79.2 (CH2), 123.4(Ar), 128.7 (Ar), 134.9 (Ar), 163.7 (CO). The residue was dissolved inCH₂ Cl₂ (140 mL) and succinic anhydride (2.6 g, 26 mmole) and DMAP (3.2g, 26.3 mmole) were added. The resulting reaction mixture was stirred atroom temperature for 12 hours under an argon atmosphere. NaH₂ PO₄ (10%aq, 100 mL) was added and the stirring was continued for 15 minutes. Theaqueous layer was extracted several times with CH₂ Cl₂. The combine CH₂Cl₂ layers were dried Na₂ SO₄ and concentrated to give a yellow residue.The residue was purified by flash chromatography over silica gel usingMeOH/CH₂ Cl₂ as the eluent. The pure fractions were pooled together andevaporated to dryness to give 1.3 g (21.7%) of the title compound: TLC(Rf: 0.4; 5% MeOH/CH₂ Cl₂), ¹ H NMR (DMSO-d6) δ 2.5 (s, 4 H, succinylCH2), 4.3 (q, 4 H, CH2), 7.9 (s, 4 H, Ar), 12.2 (s, 1 H, COOH).

II-b Propyl Linker--O-Succinyl Propyl Hydroxy N-phthalimide

A 1-hydroxyl N-phthalimido-3-propanol intermediate was prepared bytreating N-hydroxy phthalimide (2.4 g, 15 mmole) in DMSO (20 mL) with3-chloro-1-propanol (0.94 g, 10 mmole) and Na₂ CO₃ (1.6 g, 15 mmole).The reaction mixture was stirred at 80° C. for 1 hours under an argonatmosphere. H₂ O (50 mL) and EtOAc (100 mL) were added and the stirringwas continued for 15 minutes. The aqueous layer was extracted severaltimes with EtOAc. The combine EtOAc layers were dried over Na₂ SO₄ andconcentrated to give 2.81 g of the title compound: TLC (Rf: 0.7; 5%MeOH/CH₂ Cl₂), ¹ H NMR (DMSO-d6) δ 1.8 (m, 2 H, CH2), 3.5 (m, 2 H, CH2),4.2 (t, 2 H, CH2), 4.5 (t, 1 H, OH), 7.8 (s, 4 H, Ar). ¹³ C NMR(DMSO-d6): δ 31.3 (CH2), 57.3 (CH2), 75.5 (CH2), 123.5 (Ar), 128.7 (Ar),135.1 (Ar), 163.7 (CO).

The propyl intermediate is treated as described above for the ethylcompound to give the O-succinyl activated compound for loading on CPG.

III. 1-(O-Succinyl)-1,4,9-triaza-8-oxa-decane-9-phthaloyl (Succinyl--NH₂--(CH₂)₂ --NH--(CH₂)₃ --O--N-phthaloyl) (108a)

A mixture of amine 107 (10 mmol), succinyl anhydride (15 mmol) and DMAP(15 mmol) is dissolved in CH₂ Cl₂ (100 mL). The reaction mixture isstirred at room temperature for 12 hours under argon atmosphere. NaH₂PO₄ (10% aq, 100 mL) is added and stirring is continued for 15 minutes.The aqueous layer is extracted several times with CH₂ Cl₂. The combinedCH₂ Cl₂ layers are dried over Na₂ SO₄ and concentrated to get a yellowresidue. The residue is purified by flash chromatography over silica gelusing Hexane/EtOAc as the eluent. The pure fractions are pooled togetherand concentrated to give the title compound (108a).

EXAMPLE 12 Loading of Succinyl Intermediates on Control Pore Glass (CPG)

I. Formation of CPG-O-succinyl Ethyl N-phthalimide

CPG (5 g), DMAP (0.1 g, 0.75 mmole) and DCC (1.6 g, 7.5 mmole) wereadded to a solution of O-succinyl ethyl N-phthalimide (from example 11above) (0.44 g, 1.5 mmole) in CH₂ Cl₂ (20 mL). The mixture was shakenfor 12 hours. The resin was collected by filtration on a sintered glassfunnel and washed with CH₂ Cl₂, MeOH and ethyl ether. The resin wasdried and the loading was measured by taking a small sample for aninhydrin test. The loading test sample was carefully weighed andtreated with 5% of methyl hydrazine in MeOH for 3 hours at roomtemperature. A quantitative ninhydrin test was carried out by using thestandard extinction coefficient (ε). The loading of the compound on CPGwas determined to be 50 μmole/g. The resin was capped with aceticanhydride for use in the first coupling reaction.

II. Formation ofCPG-1-(O-Succinyl)-1,4,9-triaza-8-oxa-decane-9-phthaloyl (CPG-NH₂--(CH₂)₂ --NH--(CH₂)₃ --O--N-phthaloyl) (108a)

CPG (5 g), DMAP (0.1 g, 0.75 mmole) and DCC (1.6 g, 7.5 mmole) wereadded to a solution of compound 108a (0.44 g, 1.5 mmole) in CH₂ Cl₂ (20mL). The mixture was shaken for 12 hours. The resin was collected byfiltration on a sintered glass funnel and washed with CH₂ Cl₂, MeOH andethyl ether. The resin was dried and the loading measured by taking asmall sample for the ninhydrin test. The loading test sample wascarefully weighed and treated with 5% of methyl hydrazine in MeOH for 3hours at room temperature. A quantitative ninhydrin test was carried outby using the standard extinction coefficient (ε). The loading of thecompound on CPG determined to be 50 μmole/g. The resin was capped withacetic anhydride for use in the first coupling reaction.

EXAMPLE 13 Loading of Intermediate on p-alkoxybenzyl Alcohol Resin

I. Preparation of Resin

p-Alkoxy benzyl alcohol resin (3.5 g, 2.45 mmole) was added to a mixtureof 4-nitrophenyl chloroformate (6.4 g, 30.8 mmole) and TEA (4.8 mL, 33.9mmole) CH₂ Cl₂ (15 mL). The mixture was shaken for 12 hours. The resinwas collected by filtration on sintered glass funnel and washed with CH₂Cl₂.

II. Submonomer Amine

II-a: Phthalimidoacetaldehyde

Phthalimidoacetaldehyde diethylacetate (5.3 g, 20 mmole) was dissolvedin chloroform (100 mL) and a trifluoroacetic acid solution (50% aqueous,80 mL) was added. The reaction mixture was stirred at room temperaturefor 12 hours. The chloroform layer was collected and dried over sodiumsulfate. The filtrate was concentrated to give 3.75 g of the titlecompound (99% yield): TLC (Rf: 0.4; 40% EtOAc/Hexane), ¹ H NMR (DMSO-d6)δ 4.6 (s, 2 H, CH2), 7.8 (m, 4 H, Ar), 9.6 (s, 1 H, CHO). ¹³ C NMR(DMSO-d6): δ 47.4 (CH2), 123.4 (Ar), 131.5 (Ar), 134.8 (Ar), 167.3 (CO),196.8 (CHO).

II-b: Coupling Reaction of Phthalimidoacetaldehyde and Resin viaReductive Amination

To the phthalimidoacetaldehyde from step II-a (0.12 g, 38.5 μmole),resin (0.076 g, 0.4 mmole), 5 N HCl (12 p, 60 μmole) and sodiumcyanoborohydride (0.001 g, 15.9 μmole) were added. The mixture wasshaken for 12 hours and a small sample was taken for the standardninhydrin test. The coupling yield was 46.6%. The coupling step wasrepeated and the loading was redetermined by the standard ninhydrintest. The final overall coupling yield was 75.9%. The resin was cappedwith acetic anhydride for use in submonomer coupling reactions.

II-c: Attachment of Submonomer

Attachment of ethylene diamine to above derivatized p-alkoxybenzylalcohol resin was effected by adding ethylene diamine (2.5 mL, 37.4mmole), TEA (6.3 mL, 45 mmole) and acetonitrile (15 mL) to the resin.The mixture was shaken for 12 hours. The resin was collected byfiltration on a sintered glass funnel and washed with CH₂ Cl₂ and DMF.The resin was dried and the loading was measured by taking a smallsample for a ninhydrin test. Quantitative ninhydrin test was carried outby using the standard extinction coefficient (ε). The loading ofethylene diamine on p-alkoxy benzyl alcohol resin was 440 μmole/g (63%coupling yield). The resin was ready to be used for the first couplingreaction.

III. Polyamine

Polyamine 107 (37.4 mmole), TEA (6.3 mL, 45 mmole) and 15 mL ofacetonitrile (15 mL) were added to the resin of step II-c. The mixturewas shaken for 12 hours. The resin was collected by filtaration on asintered glass funnel and washed with CH₂ Cl₂ and DMF. The resin wasdried and the loading was measured by taking a small sample for aninhydrin test. The quantitative ninhydrin test was carried out by usingthe standard extinction coefficient (ε). The loading of polyamine 107 onp-alkoxy benzyl alcohol resin was 440 μmole/g (63% coupling yield). Theresin was ready to be used for the first coupling reaction.

EXAMPLE 14 Preparation of Library Subsets 114a-d via Alkylation UsingBenzyl Halides

Library subsets 114a-d are prepared as per the general teachings ofExample 10 except that these subsets are synthesized via an alkylationreaction in place of the Schiff's base reduction. Aralkyl halides,corresponding to the aldehydes of employed Example 10, are use for thesealkylation reactions. In this approach, direct alkylation provides thecombinatorialized positions directly. Reduction procedures areeliminated. The halides used for introduction of the letters are benzylhalides [benzyl bromide (L₁) (Aldrich catalog #B1,790-5), 3-methylbenzylbromide (L₂) (Aldrich catalog #B8,350-9), 3-methoxybenzyl chloride (L₃)(Aldrich catalog #20,938-4, and 3-nitro-benzyl bromide (L₄) (Aldrichcatalog #22,251-80)].

Step A

t-Boc polyamine 109 (42.1 g, 125 mmol) is divided into four equal partsand each is reacted separately with benzyl bromide (L₁), 3-methylbenzylbromide (L₂), 3-methoxybenzyl bromide (L₃), or 3-nitrobenzyl bromide(L₄). The reactants can be dissolved in an organic solvent selected frommethylene chloride, dichloroethane, ethyl acetate, toluene, or methanolsuitable for the individual reactant. For each reaction 1.5-10equivalents of the benzyl bromide is employed. A equivalent amount ofbase is utilized to neutralize the liberated acid generated byalkylation. Bases such as triethyl amine, DBU, pyridines, DMAP,carbonates, bicarbonates, and sodium hydride may be effectively employedin these alkylation reactions. Reactions are allowed to proceed from1-24 hours and then evaporated to dryness under reduced pressure. Theresidues are suspended between ethyl acetate and water. The organiclayers are separated, dried (MgSO₄), and reduced to dryness underreduced pressure. The individual residues may be purified by columnchromatography as needed. This procedure provides a polyamine/oxyamine115 with position 2 combinatorialized with the four selected aromaticbenzyl bromides and corresponds to the compounds 115 prepared byreductive alkylation of Example 10.

Step B & C

Equal moles of each pure reaction residue (114a-b, ≈11.0 g, ≈25 mmoleach) is dissolved in methanol and mixed together. The solution istreated with methylhydrazine (250 mmol), heated under reflux for onehour, and evaporated under reduce pressure. The residual mixture istriturated with chloroform and filtered. The filtrate is evaporated todryness under reduced pressure and the residue purified by silica gel,flash column chromatography if needed. This will provide ≈100 mmol (≈31g) of oxyamine 116.

Step D

Mixture 116 (≈31 g, ≈100 mmol) is dissolved in ethyl acetate containing1-3% glacial acetic acid and treated withN-(3-hydroxypropionaldehyde)phthalimide (105, 110 mmol). The solution isstirred at room temperature for 1 to 24 hours before treating withNaCNBH₃ (150 mmol) and stirring at room temperature for 1 to 10 hours.The mixture is mixed with water and separated. The organic phase iswashed with saturated NaHCO₃ solution, dried (MgSO₄), and evaporatedunder reduced pressure. The residual 117 is purified by flash silica gelcolumn chromatography as needed. Approximately 100 mmol (≈50 g) of 17 isobtained.

Step E--(repeat Step A & B), Combinatorilization of Position 3, ProvidesPolyamine/Oxyamine 118

t-Boc polyamine 117 (50.6 g, 100 mmol) is divided into four equal partsand each is reacted separately with benzyl bromide (L₁), 3-methylbenzylbromide (L₂), 3-methoxybenzyl bromide (L₃), or 3-nitrobenzyl bromide(L₄) as described above. Reactions are evaporated to dryness underreduced pressure. The residues are suspended between ethyl acetate andwater. The organic layers are separated, dried (MgSO₄), and reduced todryness under reduced pressure. The individual residues may be purifiedby column chromatography as needed. Equal amounts of each residue aremixed to provide polyamine/oxyamine 118 with position 2combinatorialized with the four selected aromatic benzyl bromides (andcorresponds to 118 prepared by reductive alkylation) to provide thebenzyl moieties.

Combinatorialization is continued as described in Example 10 exceptalkylation reactions with benzyl bromides are used in place of Schiff'sbase reductive alkylations. The alkylation procedures provide librariescomparable to the libraries synthesized by reductive couplings.

EXAMPLE 15 Preparation of Library Subsets 114a-d Using Benzoic Acids andBenzoic Acid Halides

Library subsets 114a-d are prepared by utilizing substituted benzoicacid halides to acylate the primary amine and secondary and primaryoxyamines followed by reduction of the resultant amide bond to afford abenzyl moiety combinatorialized at each selected site in thepolyamine/oxyamine. Benzoyl acid chloride (L₁), 3-methylbenzoyl chloride(L₂), 3-methoxybenzoyl chloride (L₃), and 3-nitrobenzoyl chloride (L₄)]corresponding to the benzyl bromides and benzaldehydes employed in thesynthesis of Examples 10 and 14 are employed.

Library subsets 114a-d are prepared as per the general teachings ofExample 10 except that these subsets are synthesized via an acylationreaction in place of the Schiff's base reduction. Aryl acid halides areused for these alkylation reactions corresponding to benzyl aldehydesemployed in the synthesis of Example 10. In this approach, directacylation provides the combinatorialized positions directly. As inExample 14, reduction procedures are eliminated. The acid halides usedfor introduction of the letters are benzoyl chloride (L₁),3-methylbenzoyl chloride (L₂), 3-methoxybenzoyl chloride (L₃), and3-nitrobenzoyl chloride (L₄).

Step A

t-Boc polyamine 109 (42.1 g, 125 mmol) is divided into four equal partsand each is reacted separately with benzoyl chloride (L₁),3-methylbenzoyl chloride (L₂), 3-methoxybenzoyl chloride (L₃), and3-nitrobenzoyl chloride (L₄). The reactants are dissolved in an organicsolvent selected from methylene chloride, dichloroethane, ethyl acetate,toluene, or methanol appropriate for the individual reactant. For eachreaction, 1.5-10 equivalents of an acid halide is employed. A equivalentamount of base is utilized to neutralize the liberated acid generated byacylation. Bases such as triethyl amine, DBU, DMAP, pyridines,carbonates, bicarbonates, and sodium hydride may be effectively employedin these alkylation reactions. The reactions are allowed to proceed from1 to 24 hours and then evaporated to dryness under reduced pressure. Theresidues are suspended between ethyl acetate and water. The organiclayers are separated, dried (MgSO₄), and concentrated to dryness underreduced pressure. The individual residues are reduced with NaCNBH₃ orother reagents known to effective convert amide bonds to thecorresponding methylenes. The individual residues may be purified bycolumn chromatography as needed. This procedure provides apolyamine/oxyamine 115 with position 2 combinatorialized with the fourselected aromatic acid chlorides and corresponds to 115 prepared byreductive alkylation of Example 10 to provide benzenzyl moieties.

Step B & C

Equal moles of each pure reaction residue (114a-b, ≈11.0 g, ≈25 mmoleach) is dissolved in methanol and mixed together. The solution istreated with methylhydrazine (250 mmol), heated under reflux for onehour, and concentrated under reduce pressure. The residual mixture istriturated with chloroform and filtered. The filtrate is concentratedand the residue purified by silica gel, flash column chromatography asneeded. This provides ≈100 mmol (≈31 g) of oxyamine 116.

Step D

Mixture 116 (≈31 g, ≈100 mmol) is dissolved in ethyl acetate containingglacial acetic acid (1-3%) and treated withN-(3-hydroxypropionaldehyde)phthalimide (105, 110 mmol). The solution isstirred at room temperature for 1-24 hours before treating with NaCNBH₃(150 mmol) and stirring at room temperature for 1-10 hours. The mixtureis mixed with water and separated. The organic phase is washed withsaturated NaHCO₃ solution, dried (MgSO₄), and concentrated under reducedpressure. The residual 117 is purified by flash silica gel columnchromatography if needed. Approximately 100 mmol (≈50 g) of 117 isobtained.

Step E (repeat Step A & B), Combinatorilization of Position 3, toProvides Polyamine/Oxyamine 118

t-Boc polyamine 117 (50.6 g, 100 mmol) is divided into four equal partsand each is reacted separately with benzoyl chloride (L₁),3-methylbenzoyl chloride (L₂), 3-methoxybenzoyl chloride (L₃), and3-nitrobenzoyl chloride (L₄) as described above. Reactions areevaporated to dryness under reduced pressure. The residues are suspendedbetween ethyl acetate and water. The organic layers are separated, dried(MgSO₄), and reduced to dryness under reduced pressure. The individualresidues are purified by column chromatography as needed. Equal amountsof each residue are mixed to provide polyamine/oxyamine 118 withposition 2 combinatorialized with the four selected aromatic acidchlorides and corresponds to 118 prepared by either reductive alkylationor alkylation as described in Examples 10 and 14.

Combinatorialization is continued as described in Example 10 exceptacylation reactions with acyl acid chlorides are used in place ofSchiff's base reductive alkylations. Acylation procedures providelibraries comparable to libraries synthesized by reductive oralkylations. The benzoyl halides used to introduce the letters arereadily available from various commercial chemical suppliers.

EXAMPLE 16 Backbone Extenders Alternate Methods of Preparation1-(tert-Butoxycarbonyl)-9-phthaloyl-1,4,9-triaza-8-oxa-nonane(t-butoxycarbonyl-NH--(CH₂)₂ --NH--(CH₂)₃ --O--N-phthaloyl) (106)

I. Via Alkylation

Step 1--Preparation of N-[(3-bromopropyl)oxy)]phthalimide (Br(CH₂)₃--O--N-phthalimide))

A mixture of N-hydroxyphthalimide (100 mmol) and 1,3-dibromopropane (100mmol) in dry DMF and triethyl amine (110 mmol) is stirred at 20-75° C.for 1-10 hours. After filtration, the mixture is evaporated to drynessunder reduced pressure. The residue is purified by silica gelchromatography to give N-[(3-bromopropyl)oxy)]phthalimide.

Step 2

A mixture of tert-butyl-N-(2-aminoethyl) carbamate (103) (100 mmol) andN-[(3-bromopropyl)oxy]phthalimide (100 mmol) in dry DMF andtriethylamine (110 mmol) is stirred at 20 to 75° C. for 1 to 25 hours.After filtration, the mixture is evaporated to dryness under reducedpressure. The residue is distributed between water and ethyl acetate.The organic layer is dried (MgSO₄) and evaporated to dryness to yieldpolyamine 106. Purification of 106 is achieved by silica gelchromatography.

II. Extension by Acylation

Step 1--Preparation of N-[(3-propionyl chloride)oxy]-phthalimide(ClCO(CH₂)₃ --O--N-phthalimide).

A mixture of N-hydroxyphthalimide (100 mmol) and 3-bromopropinoic acid(100 mmol) in dry DMF and triethyl amine (210 mmol) is stirred at 20 to75° C. for 1 to 10 hours. After filtration, the mixture is evaporated todryness under reduced pressure and distributed between water and ethylacetate. The water layer is treated with dilute HCl and extracted withethyl acetate. The organic layer is treated with thionyl chloride (112mmol), refluxed for 1 hour, and evaporated to dryness under reducedpressure to give N-[(3-propionyl chloride)oxy]-phthalimide.

Step 2

A mixture of tert-butyl-N-(2-aminoethyl) carbamate (103) (100 mmol) andN-[(3-propionyl chloride)oxy]phthalimide (100 mmol) in dry DMF andtriethyl amine (110 mmol) is stirred at 20 to 75° C. for 1 to 25 hours.After filtration, the mixture is evaporated to dryness under reducedpressure. The residue is distributed between water and ethyl acetate.The organic layer is dried (MgSO₄) and evaporated to dryness. Theresidue is dissolved in methanol and treated with NaCNBH₃. The mixtureis stirred at room temperature for 1 to 5 hours. The mixture is filteredand the filtrate is evaporated to dryness under reduced pressure toyield polyamine 106. Purification of 106 is achieved by silica gelchromatography.

EXAMPLE 17 Backbone Extenders--Amino Acid Type

Conversion of amino acids into N-(acetaldehydo)-phthalimide(OHCCHRN-phthalimide where R is an amino acid side chain) is effected byprotection of the amino acids with the phthalimido group by treatmentwith phthalic anhydride and subsequently reduced to provide aldehydoamino acid type extenders.

EXAMPLE 18 Backbone Extenders--Oxyamino Acid Type

Oxyamino acid type extenders are prepared by conversion of alkane acidsand R-substituted alkane acids intoN-[(alkyl-R-aldehydo)oxy]phthalimide, i.e.OHC-alkyl-R--O--N-phthalimide. Substituted acids are bromonated in thealpha position and subsequently reacted with N-hydroxyphthalimide toprovide N--(HO₂ C-alkyl-R)O-phthalimide. Reduction of the acid to analdehyde function providesN--(aldehydoalkyl-substituted)--O--N-phthalimide type extenders.

EVALUATION PROCEDURE 1--Nuclease Resistance

A. Evaluation of the Resistance of Oligomeric Compounds to Serum andCytoplasmic Nucleases.

Compounds of the invention can be assessed for their resistance to serumnucleases by incubation of the compounds in media containing variousconcentrations of fetal calf serum or adult human serum. Labelledcompounds are incubated for various times, treated with protease K andthen analyzed by gel electrophoresis on 20% polyacrylamine-ureadenaturing gels and subsequent autoradiography. Autoradiograms arequantitated by laser densitometry. Based upon the location of themodified linkage and the known length of the oligomeric compounds it ispossible to determine the effect on nuclease degradation by theparticular modification. For the cytoplasmic nucleases, an HL 60 cellline can be used. A post-mitochondrial supernatant is prepared bydifferential centrifugation and the labelled compounds are incubated inthis supernatant for various times. Following the incubation, compoundsare assessed for degradation as outlined above for serum nucleolyticdegradation. Autoradiography results are quantitated and are indictiveof resistances of the compounds to serum and cytoplasmic nucleases.

B. Evaluation of the Resistance of Oligomeric Compounds to Specificendo- and exo-nucleases.

Evaluation of the resistance of the compounds of the invention tospecific nucleases (i.e., endonucleases, 3',5'-exo-, and5',3'-exonucleases) can be done to determine the exact effect of thelinkages on degradation. The compounds are incubated in defined reactionbuffers specific for various selected nucleases. Following treatment ofthe products with protease K, urea is added and analysis on 20%polyacrylamide gels containing urea is done. Gel products are visualizedby staining with Stains All reagent (Sigma Chemical Co.). Laserdensitometry is used to quantitate the extent of degradation. Theeffects of the compound's linkage are determined for specific nucleasesand compared with the results obtained from the serum and cytoplasmicsystems. As with the serum and cytoplasmic nucleases, it is expectedthat the compounds of the invention will be completely resistant toendo- and exo-nucleases.

PROCEDURE 2 Use of Combinatorial Library for PLA₂ Inhibitors

A preferred target molecule for utilizing such combinatorial techniquesis the phospholipase A₂ family. Phospholipases A₂ (PLA₂) are a family ofenzymes that hydrolyze the sn-2 ester linkage of membrane phospholipidsresulting in release of a free fatty acid and a lysophospholipid (see,Dennis, E. A., The Enzymes, Vol. 16, pp. 307-353, Boyer, P. D., ed.,Academic Press, New York, 1983). Elevated levels of type II PLA₂ arecorrelated with a number of human inflammatory diseases. The PLA₂-catalyzed reaction is the rate-limiting step in the release of a numberof pro-inflammatory mediators. Arachidonic acid, a fatty acid commonlylinked at the sn-2 position, serves as a precursor to leukotrienes,prostaglandins, lipoxins and thromboxanes. The lysophospholipid can be aprecursor to platelet-activating factor. PLA₂ is regulated bypro-inflammatory cytokines and, thus, occupies a central position in theinflammatory cascade (see, e.g., Dennis, ibid.; Glaser, et al., TiPsReviews 1992, 14, 92; and Pruzanski, et al., Inflammation 1992, 16,451). All mammalian tissues evaluated thus far have exhibited PLA₂activity. At least three different types of PLA₂ are found in humans:pancreatic (type I), synovial fluid (type II) and cytosolic. Studiessuggest that additional isoenzymes exist. Type I and type II, thesecreted forms of PLA₂, share strong similarity with phospholipasesisolated from the venom of snakes. The PLA₂ enzymes are important fornormal functions including digestion, cellular membrane remodeling andrepair, and in mediation of the inflammatory response. Both cytosolicand type II enzymes are of interest as therapeutic targets. Increasedlevels of the type II PLA₂ are correlated with a variety of inflammatorydisorders including rheumatoid arthritis, osteoarthritis, inflammatorybowel disease and septic shock, suggesting that inhibitors of thisenzyme would have therapeutic utility. Additional support for a role ofPLA₂ in promoting the pathophysiology observed in certain chronicinflammatory disorders was the observation that injection of type IIPLA₂ into the footpad of rats (Vishwanath, et al., Inflammation 1988,12, 549) or into the articular space of rabbits (Bomalaski, et al., J.Immunol. 1991, 146, 3904) produced an inflammatory response. When theprotein was denatured before injection, no inflammatory response wasproduced.

The type II PLA₂ enzyme from synovial fluid is a relatively smallmolecule (about 14 kD) and can be distinguished from type I enzymes(e.g., pancreatic) by the sequence and pattern of its disulfide bonds.Both types of enzymes require calcium for activity. The crystalstructures of secreted PLA₂ enzymes from venom and pancreatic PLA₂, withand without inhibitors, have been reported (Scott, et al., Science 1990,250, 1541). Recently, the crystal structure of PLA₂ from human synovialfluid has been solved (Wery, et al., Nature 1991, 352, 79). Thestructures clarify the role of calcium and amino acid residues incatalysis. The calcium acts as a Lewis acid to activate the scissileester carbonyl of 1,2-diacylglycerophospholipids and bind the lipid, anda His-Asp side chain dyad acts as general base catalyst to activate awater molecule nucleophile. This is consistent with the absence of anyacyl enzyme intermediates, and is also comparable to the catalyticmechanism of serine proteases. The catalytic residues and the calciumion are at the end of a deep cleft (ca. 14 Å) in the enzyme. The wallsof this cleft contact the hydrocarbon portion of the phospholipid andare composed of hydrophobic and aromatic residues. Thepositively-charged amino-terminal helix is situated above the opening ofthe hydrophobic cleft. Several lines of evidence suggest that theN-terminal portion is the interfacial binding site. (see, e.g., Achari,et al., Cold Spring Harbor Symp. Quant. Biol. 1987, 52, 441; Cho, etal., J. Biol. Chem. 1988, 263, 11237; Yang, et al., Biochem. J. 1989,262, 855; and Noel, et al., J. Am. Chem. Soc. 1990, 112, 3704).

Much work has been reported in recent years on the study of themechanism and properties of PLA₂ -catalyzed hydrolysis of phospholipids.In in vitro assays, PLA₂ displays a lag phase during which the enzymeadsorbs to the substrate bilayer and a process called interfacialactivation occurs. This activation may involve desolvation of theenzyme/lipid interface or a change in the physical state of the lipidaround the cleft opening. The evidence favoring this hypothesis comesfrom studies revealing that rapid changes in PLA₂ activity occurconcurrently with changes in the fluorescence of a membrane probe(Burack, et al., Biochemistry 1993, 32, 583). This suggests that lipidrearrangement is occurring during the interfacial activation process.PLA₂ activity is maximal around the melting temperature of the lipid,where regions of gel and liquid-crystalline lipid coexist. This is alsoconsistent with the sensitivity of PLA₂ activity to temperature and tothe composition of the substrate, both of which can lead to structurallydistinct lipid arrangements separated by a boundary region. Fluorescencemicroscopy was used to simultaneously identify the physical state of thelipid and the position of the enzyme during catalysis (Grainger, et al.,FEBS Lett. 1989, 252, 73). These studies clearly show that PLA₂ bindsexclusively at the boundary region between liquid and solid phase lipid.While the hydrolysis of the secondary ester bond of1,2-diacylglycerophospholipids catalyzed by the enzyme is relativelysimple, the mechanistic and kinetic picture is clouded by the complexityof the enzyme-substrate interaction. A remarkable characteristic of PLA₂is that maximal catalytic activity is observed on substrate that isaggregated (i.e., phospholipid above its critical micelleconcentration), while low levels of activity are observed on monomericsubstrate. As a result, competitive inhibitors of PLA₂ either have ahigh affinity for the active site of the enzyme before it binds to thesubstrate bilayer or partition into the membrane and compete for theactive site with the phospholipid substrate. Although a number ofinhibitors appear to show promising inhibition of PLA₂ in biochemicalassays (see, e.g., Yuan, et al., J. Am. Chem. Soc. 1987, 109, 8071;Lombardo, et al., J. Biol. Chem. 1985, 260, 7234; Washburn, et al., J.Biol. Chem. 1991, 266, 5042; Campbell, et al., J. Chem. Soc., Chem.Commun. 1988, 1560; and Davidson, et al., Biochem. Biophys. Res. Commun.1986, 137, 587), reports describing in vivo activity are limited (see,e.g., Miyake, et al., J. Pharmacol. Exp. Ther. 1992, 263, 1302).

In one preferred embodiment, functional groups appended to the repeatingunits of the invention are selected for their potential to interactwith, and preferably inhibit, the enzyme PLA₂. Thus, the compounds ofthe invention can be used for topical and/or systematic treatment ofinflammatory diseases including atopic dermatitis and inflammatory boweldisease. In selecting the functional groups, advantage can be taken ofPLA₂ 's preference for anionic vesicles over zwitter-ionic vesicles.

Certain compounds of the invention include aromatic functional groups tofacilitate binding td the cleft of the PLA₂ enzyme. (see, Oinuma, etal., J. Med. Chem. 1991, 34, 2260; Marki, et al., Agents Actions 1993,38, 202; and Tanaka, et al., J. Antibiotics 1992, 45, 1071). Benzyl and4-hexylbenzyl groups are preferred aromatic groups. The compounds of theinvention can further include hydrophobic functional groups such astetraethylene glycol groups. Since the PLA₂ enzyme has a hydrophobicchannel, hydrophobicity is believed to be an important property ofinhibitors of the enzyme.

The PLA₂ assay can be effected using a combinatorial screening strategysuch as the SURF strategy. For this assay, the oligomer libraries arescreened for inhibition of human type II PLA₂ enzymatic activity.Typically, these libraries contain hundreds or thousands of differentcompounds. Successive iterations of the SURF technique is effected toselect unique oligomers from the library. The libraries additionally canbe screened in other in vitro assays to determine further mechanisms ofinhibition.

To maximize the identification of a tight binding oligomeric inhibitorof PLA₂ via a combinatorial approach, an array of functional groupstypically are included in a randomized library. The oligomers areassembled as described above.

STEP 1 PLA₂ Assay

The oligomer libraries are screened for inhibition of PLA₂ in an assayusing E. coli labeled with ³ H-oleic acid (see, Franson, et al., J.Lipid Res. 1974, 15, 380; and Davidson, et al., J. Biol. Chem. 1987,262, 1698) as the substrate. Type II PLA₂ (originally isolated fromsynovial fluid), expressed in a baculovirus system and partiallypurified, serves as a source of the enzyme. A series of dilutions ofeach the oligomeric pools is done in water: 10 μl of each oligomer isincubated for 5 minutes at room temperature with a mixture of 10 μlPLA₂, 20 μl 5×PLA₂ Buffer (500 mM Tris 7.0-7.5, 5 mM CaCl₂), and 50 μlwater. Each of the oligomer samples is run in duplicate. At this point,10 μl of ³ H E. coli cells is added. This mixture is incubated at 37° C.for 15 minutes. The enzymatic reaction is stopped with the addition of50 μl 2M HCL and 50 μl fatty-acid-free BSA (20 mg/ml PBS), vortexed for5 seconds, and centrifuged at high speed for 5 minutes. 165 μl of eachsupernate is then put into a scintillation vial containing 6 ml ofscintillant (ScintiVerse) and cpms are measured in a Beckman LiquidScintillation Counter. As a control, a reaction without oligomer is runalongside the other reactions as well as a baseline reaction containingno oligo as well as no PLA₂ enzyme. CPMs are corrected for bysubtracting the baseline from each reaction data point.

STEP 2 Verification of Assay

The PLA₂ test system was verified using phosphorothioateoligonucleotides with one or more strings of guanosine nucleotides (atleast 3 per string). Libraries of these oligo-nucleotides weredeconvoluted using the SURFs screening strategy and were shown to havean inhibitory effect on the PLA₂ enzyme. Knowing that phosphorothioateoligonucleotides inhibit PLA₂ with some sequence specificity, an eightnucleotide phosphorothioate library consisting of the four natural baseswas used to test the assay system for suitability as a SURF screen. Thislibrary had been synthesized for use in another system and all subsetswere not still available (indicated by dashes in Table III, below).Using the SURF method, it was confirmed that a stretch of guanosineswere necessary for inhibition of PLA₂ activity by the phosphorothioateoligonucleotide (Table III, below).

The assay was sensitive and accurate enough to discriminate betweensubsets of oligomers so that an inhibitory sequence could be selected.In each of the first three rounds of selection, the most active subsetwas readily determined. After 5 rounds, there was little difference inthe activity of the subsets with at least 5 G's in a row, suggestingthat the terminal positions are not critical for the inhibitoryactivity. The IC₅₀ of the "winner" improves (enzyme activity decreases)as more of the positions are fixed. As a test of the reproducibility ofthe assay, an eight nucleotide phosphorothioate oligonucleotide of asingle sequence (TTGGGGTT) was assayed with each round of testing. Thisoligonucleotide acted as an internal control of the accuracy of theassay; the IC₅₀ was 8 μM in each assay.

                  TABLE                                                           ______________________________________                                        Inhibition of PLA.sub.2 Activity by Library                                               Subsets 1C.sub.50 (mM)                                            Subsets     X = A   X = G     X = C X = T                                     ______________________________________                                        Round 2                                                                         NNGNXNNN >50 25 >50 >50                                                       Round 3                                                                       NNGXGNNN --  10 >50 --                                                        Round 4                                                                       NNGGGXNN 9 4 6 18                                                             Round 5                                                                       NAGGGGXN 4 2 4 4                                                              NGGGGGXN 2.5 2 3 3                                                            NCGGGGXN 5 4 5 5                                                              NTGGGGXN 19 5 17 15                                                         ______________________________________                                    

STEP 3 Assay of Library of Oligomeric Compounds Against PLA₂

The set of compounds 122a-d constituting a library as prepared bygeneral procedure Example 8 above, is tested in the PLA₂ assay foridentification of inhibitors of type II PLA₂. Confirmation of the"winners" is made to confirm that the oligomers binds to enzyme ratherthan substrate and that the inhibition of any oligomer selected isspecific for type II PLA₂. An assay using ¹⁴ C-phosphatidyl ethanolamine(¹⁴ C-PE) as substrate, rather than E. coli membrane, is used to insureenzyme rather than substrate specificity. Micelles of ¹⁴ C-PE anddeoxycholate are incubated with the enzyme and oligomer. ¹⁴ C-labeledarachidonic acid released as a result of PLA₂ -catalyzed hydrolysis isseparated from substrate by thin layer chromatography and theradioactive product is quantitated. The "winner" is compared tophosphatidyl ethanolamine, the preferred substrate of human type IIPLA₂, to confirm its activity. PLA₂ from other sources (snake venom,pancreatic, bee venom) and phospholipase C, phospholipase D andlysophospholipase can be used to further confirm that the inhibition isspecific for human type II PLA₂.

PROCEDURE 3 Probe for the Detection of Specific mRNA in BiologicalSample

For the reliable, rapid, simultaneous quantification of multiplevarieties of mRNA in a biological sample without the need to purify themRNA from other cellular components, a mRNA of interest from a suitablebiological sample, i.e., mRNA of a blood borne virus, a bacterialpathogen product in stool, urine and other like biological samples, isidentified using standard microbiological techniques. An oligomericcompound of the invention having "nucleobase" functional groups(adenine, guanine, thymine and cytosine as the letters) complementary tothe nucleic acid sequence of this mRNA is prepared as per the aboveexamples. The oligomeric compound is immobilized on insoluble CPG solidsupport utilizing the procedure of Pon, R. T., Protocols forOligonucleotides and Analogs, Agrawal, S., Ed., Humana Press, Totowa,N.J., 1993, p 465-496. A known aliquot of the biological sample underinvestigation is incubated with the insoluble CPG support having theoligomer thereon for a time sufficient to hybridize the mRNA to oligomerand thus to link the mRNA via the oligomer to the solid support. Thisimmobilizes mRNA present in the sample to the CPG support. Othernon-immobilized materials and components are then washed off the CPGwith a wash media suitable for use with the biological sample. The mRNAon the support is labelled with ethidium bromide, biotin or a commercialradionucleotide and the amount of label immobilized on the CPG supportis measured to indicate the amount of mRNA present in the biologicalsample.

PROCEDURE 4 Leukotriene B₄ Assay

Leukotriene D₄ (LTB₄) has been implicated in a variety of humaninflammatory diseases, and its pharmacological effects are mediated viaits interaction with specific surface cell receptors. Library subsetsare screened for competitive inhibition of radiolabeled LTB₄ binding toa receptor preparation.

A Nenquest™ Drug Discovery System Kit (NEN Research Products, Boston,Mass.) is used to select an inhibitor of the interaction of LeukotrieneB₄ (LTB₄) with receptors on a preparation of guinea pig spleen membrane.[³ H] Leukotriene B₄ reagent is prepared by adding 5 mL of liganddiluent (phosphate buffer containing NaCl, MgCl₂, EDTA and Bacitracin,pH 7.2) to 0.25 mL of the radioligand. The receptor preparation is madeby lothawing the concentrate, adding 35 mL of ligand diluent andswirling gently in order to resuspend the receptor homogenously.Reagents are kept on ice during the course of the experiment, and theremaining portions are stored at -20° C.

Library subsets 122a-d prepared as per general procedure of Example 8above are diluted to 5 μM, 50 μM and 500 μM in phosphate buffer (1×PBS,0.1% azide and 0.1% BSA, pH 7.2), yielding final test concentrations of0.5 μM, 5 μM and 50 μM, respectively. Samples are assayed in duplicate.[³ H] LTB₄ (25 μL) is added to 25 μL of either appropriately dilutedstandard (unlabeled LTB₄) or library subset. The receptor suspension(0.2 mL) is added to each tube. Samples are incubated at 4° C. for 2hours. Controls include [³ H] LTB₄ without receptor suspension (totalcount vials), and sample of ligand and receptor without librarymolecules (standard).

After the incubation period, the samples are filtered through GF/B paperthat had been previously rinsed with cold saline. The contents of eachtube are aspirated onto the filter paper to remove unbound ligand fromthe membrane preparation, and the tubes washed (2×4 mL) with coldsaline. The filter paper is removed from the filtration unit and thefilter disks are placed in appropriate vials for scintillation counting.Fluor is added, and the vials shaken and allowed to stand at roomtemperature for 2 to 3 hours prior to counting. The counts/minute (cpm)obtained for each sample are subtracted from those obtained from thetotal count vials to determine the net cpm for each sample. The degreeof inhibition of binding for each library subset is determined relativeto the standard (sample of ligand and receptor without librarymolecules).

For the purpose of illustration, consider benzyl (Bn), m-methylbenzyl(MBn), m-nitrobenzyl (NBn), and m-methoxybenzyl (MoBn) as the monomerunits in position X to be used in the synthesis and selection of anoligomer with the best activity in the LTB₄ assay. The oligomer to becombinatorialized is shown below:

    L.sub.1 NH--(CH.sub.2).sub.2 --NL.sub.2 --(CH.sub.2).sub.3 O--NL.sub.3 --(CH.sub.2).sub.3 O--NL.sub.4 --(CH.sub.2).sub.3 O--NHX

Initially, four subsets of oligomer libraries are synthesized, wherein Xis one of the listed monomer units. Each subset has a fixed monomer unitat the X position which is distinct from the monomer unit present atthat position in each of the other subsets, and the other sites ofcombinatorialization, i.e. L₁, L₂, L₃, and L₄, represent an equimolarmixture of the listed monomer units (122a-d).

    L.sub.1 NH--(CH.sub.2).sub.2 --NL.sub.2 --(CH.sub.2).sub.3 O--NL.sub.3 --(CH.sub.2).sub.3 O--NL.sub.4 --(CH.sub.2).sub.3 O--NH--Bn122a

    L.sub.1 NH--(CH.sub.2).sub.2 --NL.sub.2 --(CH.sub.2).sub.3 O--NL.sub.3 --(CH.sub.2).sub.3 O--NL.sub.4 --(CH.sub.2).sub.3 O--NH--MBn122b

    L.sub.1 NH--(CH.sub.2).sub.2 --NL.sub.2 --(CH.sub.2).sub.3 O--NL.sub.3 --(CH.sub.2).sub.3 O--NL.sub.4 --(CH.sub.2).sub.3 O--NH--NBn122c

    L.sub.1 NH--(CH.sub.2).sub.2 --NL.sub.2 --(CH.sub.2).sub.3 O--NL.sub.3 --(CH.sub.2).sub.3 O--NL.sub.4 --(CH.sub.2).sub.3 O--NH--MoBn122d

Identification of the oligomer subset with the best activity leads tothe determination of the ideal monomer unit at position X in theoligomer, e.g. oligomer subset 122b. The oligomer subset 122b is thenchosen for further combinatorialization.

In the second round of deconvolution, four oligomer subsets aresynthesized wherein each subset has a different monomer unit at the L₁position in the oligomer, and the other combinatorial sites represent anequimolar mixture of the four monomer units. These subsets are assayedfor activity, and the best oligomer subset (as shown below) is chosenfor the next round of deconvolution.

    Bn--NH--(CH.sub.2).sub.2 --NL.sub.2 --(CH.sub.2).sub.3 O--NL.sub.3 --(CH.sub.2).sub.3 O--NL.sub.4 --(CH.sub.2).sub.3 O--NH--MBn

By performing subsequent rounds in this manner, the ideal monomer unit,i.e. the monomer unit that imparts greatest activity to the oligomer, isdetermined for each of the remaining sites of combinatorialization,namely L₂, L₃ and L₄. At the end of the final round of decovolution, aunique oligomer with the best activity in the LTB₄ assay is identified.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

What is claimed is:
 1. A compound having the structure: ##STR5##wherein: each Z is, independently, H, a nitrogen protecting group,--T--L, N(--T--L)₂, a reporter molecule, or an RNA cleaving group;zz isfrom 1 to about 90; each k is, independently, 0 or 1; at least one Q isO, and each remaining Q is independently O or (CH₂)_(m) ; each A is,independently, N--T--L, C(O), a single bond, or (CH₂)_(m) ; each m is,independently, from 1 to 5; each T is, independently, a single bond or agroup having structure II:

    --[CR.sup.1 R.sup.2 ].sub.n --B--[CR.sup.1 R.sup.2 ].sub.o --[D].sub.p --[N(R.sup.3)].sub.q --                                   II

where:D is C(O), C(S), C(R¹)(NR³ R⁴), CR¹ R², or NR³ ; B is a singlebond, CH═CH, C.tbd.C, O, S or NR⁴ ; each R¹ and R² is, independently,selected from the group consisting of hydrogen, alkyl having 1 to about12 carbon atoms, alkenyl having 1 to about 12 carbon atoms, hydroxy-,alkoxy- or alkylthio-substituted alkyl having 1 to about 12 carbonatoms, hydroxy-, alkoxy- or alkylthio-substituted alkenyl having 1 toabout 12 carbon atoms, hydroxy, alkoxy, alkylthio, arnino and halogen;each R³ and R⁴ are, independently, H, alkyl having 1 to about 10 carbonatoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 toabout 10 carbon atoms, aryl having 7 to about 14 carbon atoms,heterocyclic; or R³ and R⁴, together, are cycloalkyl having 3 to about10 carbon atoms or cycloalkenyl having 4 to about 10 carbon atoms; n ando are, independently, 0 to 5; q and p are, independently 0 or 1; each Lis, independently, H, substituted or unsubstituted C₂ -C₁₀ alkyl,substituted or unsubstituted C₂ -C₁₀ alkenyl, substituted orunsubstituted C₂ -C₁₀ alkynyl, substituted or unsubstituted C₄ -C₇carbocylo alkyl, substituted or unsubstituted C₄ -C₇ carbocylo alkenyl,substituted or unsubstituted C₇ -C₁₄ aralkyl, a heterocyclic moietyhaving heteroatoms selected from nitrogen, oxygen and sulfur, where saidsubstitutions are selected from alkyl, alkenyl, alkynyl, alkoxy, thiol,thioalkoxy, hydroxyl, aryl, benzyl, phenyl, nitro, and halogen, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, a metal coordination group, a conjugate group,halogen, hydroxyl, thiol, keto, carboxyl, amide, ethers, thioethers,arnidine (C(═NH)NR³ R⁴), guanidine (NHC(═NH)NR³ R⁴), glutamyl CH(NR³R⁴)(C(═O)OR³), nitrate (ONO₂), nitro (NO₂), nitrile, trifluoromethyl(--CF₃), trifluoromethoxy (--OCF₃), O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, azido (N₃),hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide (SO), sulfone (SO₂),sulfide (S--), disulfide (S--S), silyl, a nucleosidic base, an aminoacid side chain, a nitrogen protecting group, a carbohydrate, a drug, ora group capable of hydrogen bonding, provided that when each T is asingle bond at least one L is not H or alkyl.
 2. The compound of claim 1wherein:B is a single bond; o, p and q are zero; and L is C₂ -C₁₀ alkylor substituted alkyl, C₂ -C₁₀ alkenyl or substituted alkenyl, C₂ -C₁₀alkynyl or substituted alkynyl, C₄ -C₇ carbocylo alkyl or alkenyl, C₇-C₁₄ aralkyl or substituted aralkyl, and where the substituent groupsare selected from hydroxyl, alkoxy, alcohol, benzyl, phenyl, nitro,thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, or alkynyl groups;halogen; hydroxyl (OH); thiol (SH); keto (C═O); carboxyl (COOH); amide(CONR); amidine (C(═NH)NR³ R⁴); guanidine (NHC(═NH)NR³ R⁴); glutamylCH(NR³ R⁴)(C(═O)OR³); O-alkyl; S-alkyl; NH-alkyl; N-dialkyl; O-aralkyl;S-aralkyl; NH-aralkyl; amino (NH₂); a nucleosidic base; or an amino acidside chain.
 3. The compound of claim 1 wherein said zz is from 10 toabout
 20. 4. The compound of claim 1 wherein said zz is from 2 to about15.
 5. A composition comprising at least three compounds, each of saidcompounds having the formula: ##STR6## wherein: each Z is,independently, H, a nitrogen protecting group, --T--L, N(--T--L)₂, areporter molecule, or an RNA cleaving group;zz is from 1 to about 90;each k is, independently, 0 or 1; at least one Q is N--T--L or O, andeach remaining Q is independently N--T--L, O, or (CH₂)_(m) ; each A is,independently, N--T--L, C(O), a single bond, or (CH₂)_(m) ; each m is,independently, from 1 to 5; each T is, independently, a single bond or agroup having structure II:

    --[CR.sup.1 R.sup.2 ].sub.n --B--[CR.sup.1 R.sup.2 ].sub.o --[D].sub.p --[N(R.sup.3)].sub.q --                                   II

where:D is C(O), C(S), C(R¹)(NR³ R⁴), CR¹ R², or NR³ ; B is a singlebond, CH═CH, C.tbd.C, O, S or NR⁴ ; each R¹ and R² is, independently,selected from the group consisting of hydrogen, alkyl having 1 to about12 carbon atoms, alkenyl having 1 to about 12 carbon atoms, hydroxy-,alkoxy- or alkylthio-substituted alkyl or alkenyl having 1 to about 12carbon atoms, hydroxy, alkoxy, alkylthio, amino and halogen; each R³ andR⁴ are, independently, H, alkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 7 to about 14 carbon atoms, heterocyclic; orR³ and R⁴, together, are cycloalkyl having 3 to about 10 carbon atoms orcycloalkenyl having 4 to about 10 carbon atoms; n and o are,independently, 0 to 5; q and p are, independently 0 or 1; each L is,independently, H, substituted or unsubstituted C₂ -C₁₀ alkyl,substituted or unsubstituted C₂ -C₁₀ alkenyl, substituted orunsubstituted C₂ -C₁₀ alkynyl, substituted or unsubstituted C₄ -C₇carbocylo alkyl, substituted or unsubstituted C₄ -C₇ carbocylo alkenyl,substituted or unsubstituted C₇ -C₁₄ aralkyl, a heterocyclic moietyhaving heteroatoms selected from nitrogen, oxygen and sulfur, where saidsubstitutions are selected from alkyl, alkenyl, alkynyl, alkoxy, thiol,thioalkoxy, hydroxyl, aryl, benzyl, phenyl, nitro, and halogen, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, a metal coordination group, a conjugate group,halogen, hydroxyl, thiol, keto, carboxyl, amide, ethers, thioethers,amidine (C(═NH) NR³ R⁴), guanidine (NHC(═NH) NR³ R⁴), glutamyl CH(NR³R⁴)(C(═O)OR³), nitrate (ONO₂), nitro (NO₂), nitrile, trifluoromethyl(--CF₃), trifluoromethoxy (--OCF₃), O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, azido (N₃),hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide (SO), sulfone (SO₂),sulfide (S--), disulfide (S--S), silyl, a nucleosidic base, an aminoacid side chain, a nitrogen protecting group, a carbohydrate, a drug, ora group capable of hydrogen bonding, provided that when each T is asingle bond at least one L is not H or alkyl.
 6. The composition ofclaim 5 wherein each of said zz is from 10 to about
 40. 7. Thecomposition of claim 5 wherein each of said zz is from 10 to about 20.8. The composition of claim 5 wherein each of said zz is from 2 to about15.
 9. A compound having the structure: ##STR7## wherein: each Z is,independently, H, a nitrogen protecting group, --T--L, N(--T--L)₂, areporter molecule, or an RNA cleaving group;zz is from 1 to about 90;each k is, independently, 0 or 1; at least one Q is N--T--L or O, andeach remaining Q is independently N--T--L, O, or (CH₂)_(m) ; each A is,independently, N--T--L, C(O), a single bond, or (CH₂)_(m) ; each m is,independently, from 1 to 5; each T is, independently, a single bond or agroup having structure II:

    --[CR.sup.1 R.sup.2 ].sub.n --B--[CR.sup.1 R.sup.2 ].sub.o --[D].sub.p --[N(R.sup.3)].sub.q --                                   II

where:D is C(O), C(S), C(R¹)(NR³ R⁴), CR¹ R², or NR³ ; B is a singlebond, CH═CH, C.tbd.C, O, S or NR⁴ ; each R¹ and R² is, independently,selected from the group consisting of hydrogen, alkyl having 1 to about12 carbon atoms, alkenyl having 1 to about 12 carbon atoms, hydroxy-,alkoxy- or alkylthio-substituted alkyl having 1 to about 12 carbonatoms, hydroxy-, alkoxy-, or alkylthio-substituted alkenyl having 1 toabout 12 carbon atoms, hydroxy, alkoxy, alkylthio, amino and halogen;each R³ and R⁴ are, independently, H, alkyl having 1 to about 10 carbonatoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 toabout 10 carbon atoms, aryl having 7 to about 14 carbon atoms,heterocyclic; or R³ and R⁴, together, are cycloalkyl having 3 to about10 carbon atoms or cycloalkenyl having 4 to about 10 carbon atoms; n ando are, independently, 0 to 5; q and p are, independently 0 or 1; each Lis, independently, H, substituted or unsubstituted C₂ -C₁₀ alkyl,substituted or unsubstituted C₂ -C₁₀ alkenyl, substituted orunsubstituted C₂ -C₁₀ alkynyl, substituted or unsubstituted C₄ -C₇carbocylo alkyl, substituted or unsubstituted C₄ -C₇ carbocylo alkenyl,substituted or unsubstituted C₇ -C₁₄ aralkyl, a heterocyclic moietyhaving heteroatoms selected from nitrogen, oxygen and sulfur, where saidsubstitutions are selected from alkyl, alkenyl, alkynyl, alkoxy, thiol,thioalkoxy, hydroxyl, aryl, benzyl, phenyl, nitro, and halogen, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, a metal coordination group, a conjugate group,halogen, hydroxyl, thiol, keto, carboxyl, amide, ethers, thioethers,amidine (C(═NH)NR³ R⁴), guanidine (NHC(═NH)NR³ R⁴), glutamyl CH(NR³R⁴)(C(═O)OR³), nitrate (ONO₂), nitro (NO₂), nitrile, trifluoromethyl(--CF₃), trifluoromethoxy (--OCF₃), O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, azido (N₃),hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide (SO), sulfone (SO₂),sulfide (S--), disulfide (S--S), silyl, a nucleosidic base, an aminoacid side chain, a nitrogen protecting group, a carbohydrate, a drug, ora group capable of hydrogen bonding, provided that when each T is asingle bond at least one L is not H or alkyl; and wherein T--L is C₂ C₁₀alkyl or substituted alkyl, C₂ -C₁₀ alkenyl or substituted alkenyl, C₂-C₁₀ alkynyl or substituted alkynyl, C₄ -C₇ carbocylo alkyl or alkenyl,or C₇ -C₁₄ aralkyl or substituted aralkyl.
 10. A compound having thestructure: ##STR8## wherein: each Z is, independently, H, a nitrogenprotecting group, --T--L, N(--T--L)₂, a reporter molecule, or an RNAcleaving group;zz is from 1 to about 90; each k is, independently, 0 or1; at least one Q is N--T--L or O, and each remaining Q is independentlyN--T--L, O, or (CH₂)_(m) ; each A is, independently, (CH₂)_(m) ; each mis, independently, from 1 to 5; each T is, independently, a single bondor a group having structure II:

    --[CR.sup.1 R.sup.2 ].sub.n --B--[CR.sup.1 R.sup.2 ].sub.o --[D].sub.p --[N(R.sup.3)].sub.q --                                   II

where:D is C(O), C(S), C(R¹)(NR³ R⁴), CR¹ R², or NR³ ; B is a singlebond, CH═CH, C.tbd.C, O, S or NR⁴ ; each R¹ and R² is, independently,selected from the group consisting of hydrogen, alkyl having 1 to about12 carbon atoms, alkenyl having 1 to about 12 carbon atoms, hydroxy-,alkoxy- or alkylthio-substituted alkyl having 1 to about 12 carbonatoms, hydroxy-, alkoxy-, or alkylthio-substituted alkenyl having 1 toabout 12 carbon atoms, hydroxy, alkoxy, alkylthio, amino and halogen;each R³ and R⁴ are, independently, H, alkyl having 1 to about 10 carbonatoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 toabout 10 carbon atoms, aryl having 7 to about 14 carbon atoms,heterocyclic; or R³ and R⁴, together, are cycloalkyl having 3 to about10 carbon atoms or cycloalkenyl having 4 to about 10 carbon atoms; n ando are, independently, 0 to 5; q and p are, independently 0 or 1; each Lis, independently, H, substituted or unsubstituted C₂ -C₁₀ alkyl,substituted or unsubstituted C₂ -C₁₀ alkenyl, substitutedorunsubstituted C₂ -C₁₀ alkynyl, substituted or unsubstituted C₄ -C₇carbocylo alkyl, substituted or unsubstituted C₄ -C₇ carbocylo alkenyl,substituted or unsubstituted C₇ -C₁₄ aralkyl, a heterocyclic moietyhaving heteroatoms selected from nitrogen, oxygen and sulfur, where saidsubstitutions are selected from alkyl, alkenyl, alkynyl, alkoxy, thiol,thioalkoxy, hydroxyl, aryl, benzyl, phenyl, nitro, and halogen, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms,apolyalkyl glycol, a metal coordination group, a conjugate group,halogen, hydroxyl, thiol, keto, carboxyl, amide, ethers, thioethers,amidine (C(═NH)NR³ R⁴), guanidine (NHC(═NH)NR³ R⁴), glutamyl CH(N³R⁴)(C(═O)OR³), nitrate (ONO₂), nitro (NO₂), nitrile, trifluoromethyl(--CF₃), trifluoromethoxy (--OCF₃), O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, azido (N₃),hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide (SO), sulfone (SO₂),sulfide (S--), disulfide (S--S), silyl, a nucleosidic base, an aminoacid side chain, a nitrogen protecting group, a carbohydrate, a drug, ora group capable of hydrogen bonding, provided that when each T is asingle bond at least one L is not H or alkyl.
 11. A compound having thestructure: ##STR9## wherein: each Z is, independently, H, a nitrogenprotecting group, --T--L,N(--T--L)₂, a reporter molecule, or an RNAcleaving group;zz is from 1 to about 90; each k is, independently, 0 or1; at least one Q is N--T--L or O, and each remaining Q is independentlyN--T--L, O, or (CH₂)_(m) ; at least one Q is N--T--L or O, and eachremaining Q is independently N--T--L, O, or (CH₂)_(m) ; each A is,independently, N--T--L, C(O), a single bond, or (CH₂)_(m) ; each m is,independently, from 1 to 5; each T is, independently, a single bond or agroup having structure II:

    --[CR.sup.1 R.sup.2 ].sub.n --B--[CR.sup.1 R.sup.2 ].sub.o --[D].sub.p --[N(R.sup.3)].sub.q --                                   II

where:D is C(O), C(S), C(R¹)(NR³ R⁴), CR¹ R², or NR³ ; B is a singlebond, CH═CH, C.tbd.C, O, S or NR⁴ ; each R¹ and R² is, independently,selected from the group consisting of hydrogen, alkyl having 1 to about12 carbon atoms, alkenyl having 1 to about 12 carbon atoms, hydroxy-,alkoxy- or alkylthio-substituted alkyl having 1 to about 12 carbonatoms, hydroxy-, alkoxy-, or alkylthio-substituted alkenyl having 1 toabout 12 carbon atoms, hydroxy, alkoxy, alkylthio, amino and halogen;each R³ and R⁴ are, independently, H, alkyl having 1 to about 10 carbonatoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 toabout 10 carbon atoms, aryl having 7 to about 14 carbon atoms,heterocyclic; or R³ and R⁴, together, are cycloalkyl having 3 to about10 carbon atoms or cycloalkenyl having 4 to about 10 carbon atoms; n ando are, independently, 0 to 5; q and p are, independently 0 or 1; each Lis, independently, H, substituted or unsubstituted C₂ -C₁₀ alkyl,substituted or unsubstituted C₂ -C₁₀ alkenyl, substitutedorunsubstituted C₂ -C₁₀ alkynyl, substituted or unsubstituted C₄ -C₇carbocylo alkyl, substituted or unsubstituted C₄ -C₇ carbocylo alkenyl,substituted or unsubstituted C₇ -C₁₄ aralkyl, a heterocyclic moietyhaving heteroatoms selected from nitrogen, oxygen and sulfur, where saidsubstitutions are selected from alkyl, alkenyl, alkynyl, alkoxy, thiol,thioalkoxy, hydroxyl, aryl, benzyl, phenyl, nitro, and halogen, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, a metal coordination group, a conjugate group,halogen, hydroxyl, thiol, keto, carboxyl, amide, ethers, thioethers,amidine (C(═NH)NR³ R⁴), guanidine (NHC(═NH)NR³ R⁴), glutamyl CH(NR³R⁴)(C(═O)OR³), nitrate (ONO₂), nitro (NO₂), nitrile, trifluoromethyl(--CF₃), trifluoromethoxy (--OCF₃), O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, azido (N₃),hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide (SO), sulfone (SO₂),sulfide (S--), disulfide (S--S), silyl, a nucleosidic base, an aminoacid side chain, a nitrogen protecting group, a carbohydrate, a drug, ora group capable of hydrogen bonding, provided that when each T is asingle bond at least one L is not H or alkyl; and wherein --T--L is atert-butoxycarbonyl, sulfenyltriphenyl or phthaloyl nitrogen protectinggroup.
 12. A compound having the structure: ##STR10## wherein: each Zis, independently, H, a nitrogen protecting group, --T--L, N(--T--L)₂, areporter molecule, or an RNA cleaving group;zz is from 1 to about 90;each k is, independently, 0 or 1; at least one Q is N--T--L or O, andeach remaining Q is independently N--T--L, O, or (CH₂)_(m) ; each A is,independently, N--T--L, C(O), (CH₂)_(m) ; each m is, independently, from1 to 5; each T is, independently, a single bond or a group havingstructure II:

    --[CR.sup.1 R.sup.2 ].sub.n --B--[CR.sup.1 R.sup.2 ].sub.o --[D].sub.p --[N(R.sup.3)].sub.q --                                   II

where:D is C(O), C(S), C(R¹)(NR³ R⁴), CR¹ R², or NR³ ; B is a singlebond, CH═CH, C.tbd.C, O, S or NR⁴ ; each R¹ and R² is, independently,selected from the group consisting of hydrogen, alkyl having 1 to about12 carbon atoms, alkenyl having 1 to about 12 carbon atoms, hydroxy-,alkoxy- or alkylthio-substituted alkyl having 1 to about 12 carbonatoms, hydroxy-, alkoxy-, or alkylthio-substituted alkenyl having 1 toabout 12 carbon atoms, hydroxy, alkoxy, alkylthio, amino and halogen;each R³ and R⁴ are, independently, H, alkyl having 1 to about 10 carbonatoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 toabout 10 carbon atoms, aryl having 7 to about 14 carbon atoms,heterocyclic; or R³ and R⁴, together, are cycloalkyl having 3 to about10 carbon atoms or cycloalkenyl having 4 to about 10 carbon atoms; n ando are, independently, 0 to 5; q and p are, independently 0 or 1; each Lis, independently, H, substituted or unsubstituted C₂ -C₁₀ alkyl,substituted or unsubstituted C₂ -C₁₀ alkenyl, substitutedorunsubstituted C₂ -C₁₀ alkynyl, substituted or unsubstituted C₄ -C₇carbocylo alkyl, substituted or unsubstituted C₄ -C₇ carbocylo alkenyl,substituted or unsubstituted C₇ -C₁₄ aralkyl, a heterocyclic moietyhaving heteroatoms selected from nitrogen, oxygen and sulfur, where saidsubstitutions are selected from alkyl, alkenyl, alkynyl, alkoxy, thiol,thioalkoxy, hydroxyl, aryl, benzyl, phenyl, nitro, and halogen, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, a metal coordination group, a conjugate group,halogen, hydroxyl, thiol, keto, carboxyl, amide, ethers, thioethers,amidine (C(═NH)NR³ R⁴), guanidine (NHC(═NH)NR³ R⁴), glutamyl CH(NR³R⁴)(C(═O)OR³), nitrate (ONO₂), nitro (NO₂), nitrile, trifluoromethyl(--CF₃), trifluoromethoxy (--OCF₃), O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, azido (N₃),hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide (SO), sulfone (SO₂),sulfide (S--), disulfide (S--S), silyl, a nucleosidic base, an aminoacid side chain, a nitrogen protecting group, a carbohydrate, a drug, ora group capable of hydrogen bonding, provided that when each T is asingle bond at least one L is not H or alkyl; and wherein at least oneof said Q is O.
 13. A compound having the structure: ##STR11## wherein:each Z is, independently, H, a nitrogen protecting group, --T--L,N(--T--L)₂, a reporter molecule, or an RNA cleaving group;zz is from 1to about 90; each k is, independently, 0 or 1; at least one Q is O, andeach remaining Q is independently O or (CH₂)_(m) ; each A is,independently, N--T--L, C(O), a single bond, or (CH₂)_(m) ; each m is,independently, from 1 to 5; each T is, independently, a single bond or agroup having structure II:

    --[CR.sup.1 R.sup.2 ].sub.n --B--[CR.sup.1 R.sup.2 ].sub.o --[D].sub.p --[N(R.sup.3)].sub.q --                                   II

where:D is C(O), C(S), C(R¹)(NR³ R⁴), CR¹ R², or NR³ ; B is a singlebond, CH═CH, C.tbd.C, O, S or NR⁴ ; each R¹ and R² is, independently,selected from the group consisting of hydrogen, alkyl having 1 to about12 carbon atoms, alkenyl having 1 to about 12 carbon atoms, hydroxy-,alkoxy- or alkylthio-substituted alkyl having 1 to about 12 carbonatoms, hydroxy-, alkoxy- or alkylthio-substituted alkenyl having 1 toabout 12 carbon atoms, hydroxy, alkoxy, alkylthio, arnino and halogen;each R³ and R⁴ are, independently, H, alkyl having 1 to about 10 carbonatoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 toabout 10 carbon atoms, aryl having 7 to about 14 carbon atoms,heterocyclic; or R³ and R⁴, together, are cycloalkyl having 3 to about10 carbon atoms or cycloalkenyl having 4 to about 10 carbon atoms; n ando are, independently, 0 to 5; q and p are, independently 0 or 1; each Lis, independently, H, substituted or unsubstituted C₂ -C₁₀ alkyl,substituted or unsubstituted C₂ -C₁₀ alkenyl, substituted orunsubstituted C₂ -C₁₀ alkynyl, substituted or unsubstituted C₄ -C₇carbocylo alkyl, substituted or unsubstituted C₄ -C₇ carbocylo alkenyl,substituted or unsubstituted C₇ -C₁₄ aralkyl, a heterocyclic moietyhaving heteroatoms selected from nitrogen, oxygen and sulfur, where saidsubstitutions are selected from alkyl, alkenyl, alkynyl, alkoxy, thiol,thioalkoxy, hydroxyl, aryl, benzyl, phenyl, nitro, and halogen, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, a metal coordination group, a conjugate group,halogen, hydroxyl, thiol, keto, carboxyl, amide, ethers, thioethers,arnidine (C(═NH)NR³ R⁴), guanidine (NHC(═NH)NR³ R⁴), glutamyl CH(NR³R⁴)(C(═O)OR³), nitrate (ONO₂), nitro (NO₂), nitrile, trifluoromethyl(--CF₃), trifluoromethoxy (--OCF₃), O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, azido (N₃),hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide (SO), sulfone (SO₂),sulfide (S--), disulfide (S--S), silyl, a nucleosidic base, an aminoacid side chain, a nitrogen protecting group, a carbohydrate, a drug, ora group capable of hydrogen bonding, provided that when each T is asingle bond at least one L is not H or alkyl; and wherein at least oneof said A is other than C(O).